Nucleic acid molecules encoding human calcium/calmodulin (CaMK) dependent kinase proteins

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifing orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that are related to the calcium/calmodulin-dependent protein kinase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Protein Kinases

Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phogphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.

The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.

The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

Calcium/calmodulin-dependent Protein Kinases

The novel human protein, and encoding gene, provided by the present invention is related to the family of calcium/calmodulin-dependent protein kinases, which are serine/threonine kinases. The protein of the present invention shows a particularly high degree of similarity to calcium/calmodulin-dependent protein kinase II (CaM II). CaM II is comprised of alpha, beta, gamma, and delta subunits. Each subunit is encoded by a separate gene and alternatively splice forms of each subunit have been found (Breen et al., Biochem. Biophys. Res. Commun. 236 (2), 473-478 (1997)). CaM II exerts important effects on hormones and neurotransmitters that utilize calcium as a second messenger. Beta-cell CaM II activity is associated with insulin secretion, and multiple isoforms of CaM II are expressed in human islets of Langerhans (Breen et al., Biochem. Biophys. Res. Commun. 236 (2), 473-478 (1997)). It has been suggested that CaM II controls activation-induced cellular differentiation, and is important for imparting antigen-dependent memory to T cells (Bui et al., Cell 100: 457-467, 2000). For a further review of CaM II, see Li et al., Cytogenet. Cell Genet. 66: 113-116, 1994.

Kinase proteins, particularly members of the calcium/calmodulin-dependent protein kinase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the calcium/calmodulin-dependent protein kinase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the calcium/calmodulin-dependent protein kinase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the kinase protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain.

FIG. 2 provides the predicted amino acid sequence of tle kinase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinase protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 16 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a kinase protein or part of a kinase protein and are related to the calcium/calmodulin-dependent protein kinase subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human kinase peptides and proteins that are related to the calcium/calmodulin-dependent protein kinase subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these kinase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known kinase proteins of the calcium/calmodulin-dependent protein kinase subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known calcium/calmodulin-dependent protein kinase family or subfamily of kinase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the kinase family of proteins and are related to the calcium/calmodulin-dependent protein kinase subfamily (protein a sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the kinase peptides of the present invention, kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the kinase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the kinase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. For example, a nucleic acid molecule encoding the kinase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell, The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcrip/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the kinase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a kinase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the kinase peptide. “Operatively linked” indicates that the kinase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of the kinase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant kinase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the kinase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50 wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the kinase peptides of the present invention as well as being encoded by the same genetic locus as the kinase peptide provided herein. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 10 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a kinase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by the same genetic locus as the kinase peptide provided herein. Genetic locus can readily be determined based on the genonic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 10 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 16 different nucleotide positions. Some of these SNPs, which are located 5′ of the ORF and in introns, may affect control/regulatory elements.

Paralogs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the kinase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the kinase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a kinase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as kinase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the kinase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a kinase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the kinase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the kinase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in kinase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Accordingly, the kinase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which tle mature kinase peptide is fused with another compound, such as a compound to increase the half-life of the kinase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature kinase peptide, such as a leader or secretory sequence or a sequence for purification of the mature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a kinase-effector protein interaction or kinase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, kinases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the calcium/calmodulin-dependent protein kinase subfamily (see Background of the Invention). The structural and functional infonnation provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain, Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to kinases that are related to members of the calcium/calmodulin-dependent protein kinase subfamily. Such assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase-related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the kinase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the kinase. Both the kinases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the kinase. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway that the kinase protein normally interacts (for example, another kinase). Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant kinases or appropriate fragments containing mutations that affect kinase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) kinase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase. Thus, the soluble polypeptide that competes with the target kinase region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the kinase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a kinase-binding protein and a candidate compound are incubated in the kinase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the kinase protein target molecule, or which are reactive with kinase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. These methods of treatment include the steps of administering a modulator of kinase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the kinase and are involved in kinase activity. Such kinase-binding proteins are also likely to be involved in the propagation of signals by the kinase proteins or kinase targets as, for example, downstream elements of a kinase-mediated signaling pathway. Alternatively, such kinase-binding proteins are likely to be kinase inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the kinase protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specific antibody, or a kinase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The kinase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the kinase protein in which one or more of the kinase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and kinase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or kinase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerydirin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the kinase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a kinase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the kinase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3 both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the kinase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the kinase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3, Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 10 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 16 different nucleotide positions. Some of these SNPs, which are located 5′ of the ORF and in introns, may affect control/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45 C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 16 different nucleotide positions.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determing the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 10 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the kinase gene, particularly biological and pathological processes that are mediated by the kinase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain. The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the kinase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of kinase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of kinase mRNA in the presence of the candidate compound is compared to the level of expression of kinase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), duodenal adenocarcinoma (small intestine), and fetal brain.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the kinase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in kinase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in kinase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the kinase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the kinase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 16 different nucleotide positions. Some of these SNPs, which are located 5′ of the ORF and in introns, may affect control/regulatory elements. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 10 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant kinase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenornic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 16 different nucleotide positions. Some of these SNPs, which are located 5′ of the ORF and in introns, may affect control/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control kinase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of kinase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of kinase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired kinase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the kinase protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in kinase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired kinase protein to treat the individual.

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in the placenta, breast (including mammary adenocarcinoma), skin melanotic melanoma, ovary adenocarcinoma, uterus leiomyosarcoma, Burkitt's lymphoma (lymph), and duodenal adenocarcinoma (small intestine), as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting kinase nucleic acid in a biological sample; means for determining the amount of kinase nucleic acid in the sample; and means for comparing the amount of kinase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the fall length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic ingtruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the kinase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the kinase gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 16 different nucleotide positions. Some of these SNPs, which are located 5′ of the ORF and in introns, may affect control/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified kinase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be usefull to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed; Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with kinases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a kinase protein or peptide that can be further purified to produce desired amounts of kinase protein or fragments. Thus, host cells acontaining expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the kinase protein or kinase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native kinase protein is useful for assaying compounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant kinase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native kinase protein.

Genetically engineered host cells can be farther used to produce non-human transgenic animals. A fransgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a kinase protein and identifying and evaluating modulators of kinase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the kinase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the kinase protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, kinase protein activation, and signal transduction, may not be evident from a in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo kinase protein function, including substrate interaction, the effect of specific mutant kinase proteins on kinase protein function and substrate interaction, and the effect of chimeric kinase proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more kinase protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

4 1 2061 DNA Human 1 cggtgctgcc gggctcagcc ccgtctcctc ctcttgctcc ctcggccggg cggcggtgac 60 tgtgcaccga cgtcggcgcg ggctgcaccg ccgcgtccgc ccgcccgcca gcatggccac 120 caccgccacc tgcacccgtt tcaccgacga ctaccagctc ttcgaggagc ttggcaaggg 180 tgctttctct gtggtccgca ggtgtgtgaa gaaaacctcc acgcaggagt acgcagcaaa 240 aatcatcaat accaagaaat tgtctgcccg ggatcaccag aaactagaac gtgaggctcg 300 gatatgtcga cttctgaaac atccaaacat cgtgcgcctc catgacagta tttctgaaga 360 agggtttcac tacctcgtgt ttgaccttgt taccggcggg gagctgtttg aagacattgt 420 ggccagagag tactacagtg aagcagatgc cagccactgt atacatcaga ttctggagag 480 tgttaaccac atccaccagc atgacatcgt ccacagggac ctgaagcctg agaacctgct 540 gctggcgagt aaatgcaagg gtgccgccgt caagctggct gattttggcc tagccatcga 600 agtacaggga gagcagcagg cttggtttgg ttttgctggc accccaggtt acttgtcccc 660 tgaggtcttg aggaaagatc cctatggaaa acctgtggat atctgggcct gcggggtcat 720 cctgtatatc ctcctggtgg gctatcctcc cttctgggat gaggatcagc acaagctgta 780 tcagcagatc aaggctggag cctatgattt cccatcacca gaatgggaca cggtaactcc 840 tgaagccaag aacttgatca accagatgct gaccataaac ccagcaaagc gcatcacggc 900 tgaccaggct ctcaagcacc cgtgggtctg tcaacgatcc acggtggcat ccatgatgca 960 tcgtcaggag actgtggagt gtttgcgcaa gttcaatgcc cggagaaaac tgaagggtgc 1020 catcctcacg accatgcttg tctccaggaa cttctcagtt ggcaggcaga gctccgcccc 1080 cgcctcgcct gccgcgagcg ccgccggcct ggccgggcaa gctgccaaaa gcctattgaa 1140 caagaagtcg gatggcggtg tcaagaaaag gaagtcgagt tccagcgtgc acctaatgga 1200 gccacaaacc actgtggtac acaacgctac agatgggatc aagggctcca cagagagctg 1260 caacaccacc acagaagatg aggacctcaa agctgccccg ctccgcactg ggaatggcag 1320 ctcggtgcct gaaggacgga gctcccggga cagaacagcc ccctctgcag gcatgcagcc 1380 ccagccttct ctctgctcct cagccatgcg aaaacaggag atcattaaga ttacagaaca 1440 gctgattgaa gccatcaaca atggggactt tgaggcctac acgaagattt gtgatccagg 1500 cctcacttcc tttgagcctg aggcccttgg taacctcgtg gaggggatgg atttccataa 1560 gttttacttt gagaatctcc tgtccaagaa cagcaagcct atccatacca ccatcctaaa 1620 cccacacgtc cacgtgattg gggaggacgc agcgtgcatc gcctacatcc gcctcaccca 1680 gtacatcgac gggcagggtc ggcctcgcac cagccagtca gaagagaccc gggtctggca 1740 ccgtcgggat ggcaagtggc tcaatgtcca ctatcactgc tcaggggccc ctgccgcacc 1800 gctgcagtga gctcagccac aggggcttta ggagattcca gccggaggtc caaccttcgc 1860 agccagtggc tctggagggc ctgagtgaca gcggcagtcc tgtttgtttg aggtttaaaa 1920 caattcaatt acaaaagcgg cagcagccaa tgcacgcccc tgcatgcagc cctcccgccc 1980 gcccttcgtg tctgtctctg ctgtaccgag gtgtttttta catttaagaa aaaaaaaaaa 2040 aaaaaaaaaa aaaaaaaaaa a 2061 2 565 PRT Human 2 Met Ala Thr Thr Ala Thr Cys Thr Arg Phe Thr Asp Asp Tyr Gln Leu 1 5 10 15 Phe Glu Glu Leu Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Val 20 25 30 Lys Lys Thr Ser Thr Gln Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys 35 40 45 Lys Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile 50 55 60 Cys Arg Leu Leu Lys His Pro Asn Ile Val Arg Leu His Asp Ser Ile 65 70 75 80 Ser Glu Glu Gly Phe His Tyr Leu Val Phe Asp Leu Val Thr Gly Gly 85 90 95 Glu Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp 100 105 110 Ala Ser His Cys Ile His Gln Ile Leu Glu Ser Val Asn His Ile His 115 120 125 Gln His Asp Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu 130 135 140 Ala Ser Lys Cys Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu 145 150 155 160 Ala Ile Glu Val Gln Gly Glu Gln Gln Ala Trp Phe Gly Phe Ala Gly 165 170 175 Thr Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Asp Pro Tyr Gly 180 185 190 Lys Pro Val Asp Ile Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu 195 200 205 Val Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Lys Leu Tyr Gln 210 215 220 Gln Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr 225 230 235 240 Val Thr Pro Glu Ala Lys Asn Leu Ile Asn Gln Met Leu Thr Ile Asn 245 250 255 Pro Ala Lys Arg Ile Thr Ala Asp Gln Ala Leu Lys His Pro Trp Val 260 265 270 Cys Gln Arg Ser Thr Val Ala Ser Met Met His Arg Gln Glu Thr Val 275 280 285 Glu Cys Leu Arg Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile 290 295 300 Leu Thr Thr Met Leu Val Ser Arg Asn Phe Ser Val Gly Arg Gln Ser 305 310 315 320 Ser Ala Pro Ala Ser Pro Ala Ala Ser Ala Ala Gly Leu Ala Gly Gln 325 330 335 Ala Ala Lys Ser Leu Leu Asn Lys Lys Ser Asp Gly Gly Val Lys Lys 340 345 350 Arg Lys Ser Ser Ser Ser Val His Leu Met Glu Pro Gln Thr Thr Val 355 360 365 Val His Asn Ala Thr Asp Gly Ile Lys Gly Ser Thr Glu Ser Cys Asn 370 375 380 Thr Thr Thr Glu Asp Glu Asp Leu Lys Ala Ala Pro Leu Arg Thr Gly 385 390 395 400 Asn Gly Ser Ser Val Pro Glu Gly Arg Ser Ser Arg Asp Arg Thr Ala 405 410 415 Pro Ser Ala Gly Met Gln Pro Gln Pro Ser Leu Cys Ser Ser Ala Met 420 425 430 Arg Lys Gln Glu Ile Ile Lys Ile Thr Glu Gln Leu Ile Glu Ala Ile 435 440 445 Asn Asn Gly Asp Phe Glu Ala Tyr Thr Lys Ile Cys Asp Pro Gly Leu 450 455 460 Thr Ser Phe Glu Pro Glu Ala Leu Gly Asn Leu Val Glu Gly Met Asp 465 470 475 480 Phe His Lys Phe Tyr Phe Glu Asn Leu Leu Ser Lys Asn Ser Lys Pro 485 490 495 Ile His Thr Thr Ile Leu Asn Pro His Val His Val Ile Gly Glu Asp 500 505 510 Ala Ala Cys Ile Ala Tyr Ile Arg Leu Thr Gln Tyr Ile Asp Gly Gln 515 520 525 Gly Arg Pro Arg Thr Ser Gln Ser Glu Glu Thr Arg Val Trp His Arg 530 535 540 Arg Asp Gly Lys Trp Leu Asn Val His Tyr His Cys Ser Gly Ala Pro 545 550 555 560 Ala Ala Pro Leu Gln 565 3 62804 DNA Human misc_feature (1)...(62804) n = A,T,C or G 3 ttgcccctgg cctggtctcc ctgatcaacc cgcgcctgaa gggtttcttt ctaataatgg 60 ccctggtgct tgcgcaagtc tagactgtca gctcccagag ggaaggcggc tggcagctgg 120 ctctgcgcag gctgggggcg cctcccgggc gtgcagcctg gcacaggctc cttgaccttg 180 gctctctccc cacgtgctag gagcccggtt gggggctcgg gacccgcgtg taggacccgt 240 ccagagaggt cagtggtcca gactcctaca ctcctaacac atgcaccctc gcatgcacgt 300 tcccgagccc gcgcggggtc cgccccggga caagcccata agtcgcgaac cttccagnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnntgtaa gccaccggcg ccgggcggtc tcgacattaa 960 atttcaaaat gttttctccg gtttgtcact tgtggtttta ctatgttcaa tgggtctcac 1020 caagcaattt tgcaaaatag ttaacttatt ctctttttct tacatgactt cttgactttg 1080 agccatagtt aggaaaggtt tgctcactct cacattagag taaaatttat ccacattttc 1140 atctaggatt agtgctcatt tttttattat tatgaatatc ttcttcattt ggggtttgtt 1200 catgtatatt ccatgaacaa tggacgcggg tgcagcattt tagcatcagc tatccccttc 1260 ccatccgcaa tgagctggcc gctgcagcag ccccggcccc ccacccccac ccgcggcgcc 1320 gagcccggcc actgcagccc ccgccccgcc cgccccccca gacgtttcca gagctcagag 1380 tgcgagctcc cgtttgacgg ggacgtcaag gaaaatagca tgggaagggg agttcttgat 1440 gtctgactgt gtcctctctt cccttgctgt cagttgagcc gggatgcagt gagatgaaac 1500 cggctgtggg ggggtttgag cctcactttg ccccatggtt gagggagatt tctctttcag 1560 gggatgatac cctcttttta atctttcctt ccccgacctt cagctgttcc tgctgagaga 1620 agggcagggt ctctctgctc ccttctgccc tggttctctt ggccgggacc gcagggctgt 1680 ctgagatgca gcaggtgtgt gttttcagca tcgcccaccc gctcctgatg tgcagcctga 1740 ggtggaggct gttgccttgc ccagggactg gatgaggggg tgggagcgcg gcacgccacc 1800 cacatctgtt cagtgtcctg cggtggccgc gtccttttgc ctcatgttgg atggtggtgg 1860 tcacagcgcc ggtgtgtgtg catgtacgtg agtgtgacta gaggtctggt ggtgggagca 1920 tcatcgtccc cagacttgaa gtgtgtctgt gtcactctgc cctgctccgt gtcccagttc 1980 ttttcccctt ctccctccag gggtgctttc tctgtggtcc gcaggtgtgt gaagaaaacc 2040 tccacgcagg agtacgcagc aaaaatcatc aataccaaga aattgtctgc ccggggtgag 2100 tgttccctgt cttgacctct tcctgagggt gcctccaggg gccatggttt cttttgagga 2160 agccccagga attgggggtt gtgcgtttta gcacttggag aggagttgga atttcagact 2220 ggttggactt tgtgtcaggc tgaagccaga aaaggagttg catgggggac tggaagcgcc 2280 caggtacaaa agaatgaagg aagagatgca agtagctgca gtggccccca aaggctcaag 2340 ggagttcggt cttcagggag gtggaggata tgggggtagt gggtggtaca gaatggggag 2400 ctcttaattt ggggcatttg gagcctctcc ctttggggca gtggtggcta ctgcaggcct 2460 ttcctggtcc cttcttcacc acgggctgag ttaggatgga aatgcagtaa gtgagcagct 2520 ctgacaaagc cagcctcccc tgcccaccag gcggcagaac agactcccaa gggaagggaa 2580 tctgtaaaca tcaggggagg ctgctactgg cgagggcttc tcaggaacaa attctgccag 2640 atgaacttga ttgctttttt gatcaaatta caaagttggt ggtgcagcag cagatgtagt 2700 ctgtcctggg tggagggtga tgcctcatgg tctagaaatc ccaaaggccc ggtttgggca 2760 ggaactgcac tgcctccgaa ctgcactgcc tccgagtctg aggagcataa aggccaaggc 2820 cttggggcct cacttgcgag atcctcccaa gtacctgagg cttggagggt cagggcctgt 2880 ctttcacacc ttgaacctac actctctgaa cttcctattg ggtacttgcc aaactcacct 2940 catctgatag gtgtagaccc agcaatgtgt gaagtgctct gggaacaggt ctggtgagta 3000 cagaggtcag atctcggagg gctgcagggt gcagctgggg gacaaaggtt gtgaaactca 3060 gagaaaggaa ttagggctgg gcagtaggat gccataaata tatttggagc caggacacat 3120 gccctgggga agacatgggc tttggccaat aatgacacgg gtttctctgg gataagagac 3180 ataatagatg tcccaaatgc ttagagaagc tctacaattc cacgggcttc tgtcgtgttg 3240 gcagttgttc tgggacctgt ttagcagggc cgtgtccact ccctgactgg ggactctctc 3300 tccatccctc tggtagggca ctaattgctg actcccatcc agctccatct cttgctgttc 3360 gtgtacattg cctataaagt tggacttgtt tgttttcttt ctctctgggt accttgagtc 3420 tgaggatggt tgccatagag atatgtgggc agtcagatac cctggagtgg gggtgggggg 3480 gacaacaggg gctgggctct ctggcagaca tcctctggcc aaggatggaa ggtgcaggca 3540 ggaacaatgg cttgaggctg gatacctctc ttgcccacac agcagagccc tggtgcatca 3600 gaaacagggc tggcatctgg tgtctccagt tgatgatgca atgctttgct ctcttcatct 3660 caccagtgtc ctctgaccca tgggtaagag aaggagagat ggctgggagc cgaattctgg 3720 gatgtgagga taggtgatgt ggtgacttcc tgcagctgcc tgactggggc tttcatttcc 3780 tactccttcc ctacctgcgt aaatttccat gacctgtgtg atagcctccc tttcccttcc 3840 tcacctcctt ttaaccttgt cccatctttc ccaatggata tctttccctg gccaaactgg 3900 atgagacttg atttctcgtt gatttttttt tttttcccct caagaagagg attcttgtgt 3960 aaaagtatat gcttcagaca gcaactcccc ctctcccaag atggatatgc caagactggg 4020 ctctgttgtg tggcctcatg tgccaggttg actttgggac agaggcacag atgataggca 4080 cagatgccag ccagaggggt cagaatgtgt aagtgccagc cagtactgtg tggaggtggg 4140 aaagtggaaa ggggctgtct tggagatgga gggaacaagg tggggctgga ctataggtgt 4200 gggcatggga gatgtgaact cctggagaga tctgggccag ggtagccatg ggctggttcc 4260 catggggtta gggagtgagg gccatggctt ccctgcagac tctcagttta cactatatat 4320 tttataaagg tgcagccact ggagctgggt ttcactcatc gctgtctgcc taggtctccg 4380 caggtgttgg atttctgtgt ctgggaatgt cgtgggccca ccagggtcat ctgtgaaggt 4440 ctgaaggggc ttgctgtgtt cactgggtct tcctgcctcc tgtctttctt gtttgtgatt 4500 ctctgggcta caaactgaaa agataaaaag agggtataga gctgtttctc cttggcatcc 4560 ctggtgaggt ggctaggagt cagggagagg gatcacctgt tcttctgggg gggtccaatc 4620 gagacaggaa gccttctttt gggctgttgt gtcttgtcac tgtggcctca gaggcccaca 4680 ttggcggcta ggttgcaagg tggggagttc atgcggatat gcgttgagca ctgtctttgt 4740 ctgcgggcct gtctacataa agtcactgaa agtcacataa cgtcactccg tttgcttcag 4800 aaccgtgata ggagtggagc tgggctctta agggagccca tggttccaag cttagctcca 4860 ctaggccgaa ggaggcattt aaaataggct tggatgcagg agctagtggg ccaggtgatg 4920 gcaatgataa gtcgttattt taagatttaa gagcaccccc ctcaaggagc ctgagccctt 4980 atgtcttttt ttatttttaa atcttcatat tcccttctta tctttattca tatgcataca 5040 gattttcacc tcgtggagca taacatttta tatcctgctc tctttgctta tatccaaagc 5100 atttccccca tattactaca gttgaagggc aaatggtcct ttcttctacg tcgtttagga 5160 tttatcccta aaacaatcag catcacaaga aacttctgta tatgtaccat ttatctggat 5220 tccagttgct tttaccaaga tagatactgg ggtaatgccc ttggccttac taagagatgc 5280 taccggaaac agtgttttga aatctgttat aatactttaa catatttatt taatctgtac 5340 attccgtgtg aagaaatttc ttttgaagct aaatgtaagc aaaagctttc ctctttgtga 5400 ggacctgaga ggtgagggaa gggtccttat gtgtttctat acttctgcat gggcaggccc 5460 tagcgaagtg cctgacgtat gccagccaca tacacattaa atgaatgggt caagaggact 5520 atgtaaccaa tcatggttgc cttttggctt tggctcctag gaaactcaga gtcaagttgc 5580 cagagccctt gtaccctgct acagacttgg gtcctccctt tctgatccag ggagccaagc 5640 tgcagacctg atacggctgc tggaagagag gacagatgag gataaagacc tgtgcttggg 5700 gcataaggca gagtgggaga tgtaggcaga catttagctg atgattcctc cttccctgtc 5760 actaaatggc actatagggc cactgttggg atctcttcca ggtagtgatt ttcaatttta 5820 gtgtgcgtaa ggatcaccct gagtactagt ttaaaaaata cagacttctg ggctttagcc 5880 acagagattc tgctttagga ggtctagggt ggagctgcag aatctgcatt tttaacacat 5940 gctccagtga atttcatgca ggtgaggcat gagccactct ttaagagatg ccacctaaaa 6000 tctgcaacaa cagttgctct tgccatgccc tctggaattc aacagacaca ccttggccca 6060 tccttctcca gattgtgtgt ctgccactat gtggccatct gtgcacatgg gctgttctgt 6120 gattaggggc ctcgttctgg gcctcgggat tggggtgtct gtgtctgagg ctgcggcaag 6180 ctgggtggct cgggttgtgg catgttggcc accagaaggg taaaggctgt ccctttctgg 6240 gtccagctgg ccctggggac tgaaatggga tcccctggat ggtgccagct gagagtcccc 6300 gcccccttag tgttggcctg agtagccccc atgacatttg tgtcccctgt ggtatctcca 6360 agtgagactt tcctgttaag gatctgggtg aagtgaggga aagagaaggg agggggaagc 6420 agtaatgcag ggagtgggag aaggaagaga aatccacaca gcactggaac acaggcctcg 6480 aggaagcatt taaggaggct gtgtgcgaaa ccatgctttc ctcctgagga taaaacaggc 6540 caatttctgt aaacagagaa atgggcatcc tgcatatcag tgatggagcg cctctacttt 6600 ctctcctgaa gggatggaag ccgactgcag gtccctctgt gcaaaggctt ctgccaggcg 6660 gcttttgtca cgcggtcacg ttgagctgtg ggccttagca cacacaacac tggcctgtcc 6720 ccctcccctc ccacctgtct tcctagagtg acttggggtg ctgcatcatg gtgtggggat 6780 ggaggtggga aggttgccct gtcctgtcag ggaggcccct gccttcttcc tgctgcttcc 6840 tctggtccct tgtcaccata cccttgttcg aagctgtgct gaaaccctag aggtgagtgg 6900 ctgaccccat tctctgctga gactggagat agggaagggg aggctgggtg tgaccattcc 6960 tgctcccatc tgtatgcttg ctgctctctg aacagctttg gcagaccaac aagggcctga 7020 tcccatgggt gccaaaaggg tggtgacagg aggagatggg cactttgcac ctcttgaatg 7080 cctctctgca gagccccttt gtcacctacc catggccaga cagatctgcc gcaggaccgc 7140 tggggaaatc aaagcacaaa agctttgtct ggggtctttt ttttcttttt tggttttgtg 7200 ctgcaggtgc ccatgacttt gccagggctc agacccagcg tcctcaggcc gtgtggcctc 7260 cacccactcc ttggcgcctt tctttaaaac acaggttctg gatactttgt tcctgtgatg 7320 aatcttggca tatcacctca cacctctcca tctaggcccc aagctccaag cctggtggag 7380 caaatccctc ctcgttgctg gctgaggccc cattcccgtc tgtacccacc tctctgggct 7440 gtgcggtggg gagatttcca gccactcctc cccaacacca tctccgcttc ctgggcccta 7500 tcagcagcag ccgcagcttc ccatctgctc ccctcttttc tcctcccttt ctttcccttc 7560 ccccctgctt gctgctgccc tgggaggagc tatttttagg ggctgcttcc tgggatgttt 7620 tacttggggc tggttaccat gaaggaaatg tcaccaaaac agtgggcaaa ggctgcaggc 7680 accgggagcc ctgccggggg gcatggagaa cagacggctg acccttttct ggcccttgag 7740 agcagccaga gtgcccccag gcagagcctt gccttcttgg ggcttgctag tgaccccttg 7800 gggattttct ctgtcaaagc tgattgaggg ccttttcgct atagggcatt tcttggagcc 7860 tctcgcttcc cttgccttga gatccagagg ccaaagtggg gctcaggtct ttgtgtcacc 7920 aagttaaaac tgcttgagtg agggttgaag ataaggggag gatgctgggt acatgcacag 7980 agccttgggg gttcacatgg gaccatttca ggccccgtcc ctctgtatca cagcccccag 8040 ctagtcacca ggtgtacatg tgtgagggca ttagaaacca tggtcctgct cttgtgtgtc 8100 ggatggactt tgcttttaat tggagactct ttgcatcttt agagtgagat tcaaagagga 8160 agggatgtgg catcacagtg tcagggtgag gtcggtggga tcgtggcttg ggattcccac 8220 tggtcagtgt cccaggccca gggctgtgca taagcagctg gggaaggtgg attatgacat 8280 caaatccctg cgatgtcctt gtttctgctc ctcagagtgc caaggggacc agacggcggc 8340 ctctgctgct tgggaagaag atgaaaggca ctcaggaggg cagcaagtga ggccgcctcc 8400 catggagccc tgaaatcagt ggggttgcag gaagtttctc acatccatgt ttagggtcat 8460 aggcacagac ctgcaaaata ccctttgcaa agttaagaat gtctttgaga ttggaacttg 8520 ggagagtcct cagtcagagt aggaatgtgc atcctttccc acgtacagag gattgtatgt 8580 ttacgtggca gcaggatctt atttgaagct agtgctggca tttgtgtttt ttttttagga 8640 aaatgtcact aagtcaagca ggcccatccc tgagagggcc atggagaatc tgtggccagc 8700 cctccctggc cccctgacct ggcagaggaa ggaaagggca ttggagtagg cttctgtctt 8760 caggccagag ggggaggtgg ttcaggggca ggcttggtgc accccttggc tgcaagctat 8820 cacctcccta tctgcttcct cttttctgcc tcccctggtg catctggtca cttcttgctg 8880 cccttcctgt gaaatcgtgg caccttggac caagtcctga agcacttggg cagaaggcgg 8940 gagaggttgg gtttctagga tccttgtttc ccagggcctg gctctggcct gggctcagac 9000 cactctggtc taggcaggct gctggggaaa ggctggagct gcttctgctt tctgctcctg 9060 ttgccacctc tgctaatgat ggggaaaacc tgcagagggc tgtggttgga gctgggctga 9120 aggccggcag gggtgggtct ctccatggca gtagcacaca ggcaggcagg aagtggccct 9180 gtgcaaaagc gggaagtggc agttgtcaaa caggaagggg ggggctgggc tgtgggaggg 9240 gcggggatga gcctggtaga aaggtgcgtg gaggagggtc caccttggaa ggtctgagcc 9300 tctccctagt ggttactgga aggaggggtg tctcaagggg agacaccttt gcagcacctt 9360 gagatgccga gccagggccc tcccactgtg gaccaagccc attcagtggc ctcgcccttt 9420 ttggggttgg agatgctgcg tccagctggg atgcccttgc ttttgggaaa gatgctctag 9480 aaaccactac tccatcctgg aacccctctg ctgccactgc tgctgggatg gaccctctgc 9540 ttttttgcag ccgtgggcca gccctggatg tgactacagg acaggaagtg tcaggggaag 9600 agacaggaga caacagctgg agaggctggg tggtggccgg gcagtatgtg gcagcaggaa 9660 cggggagagc ggggcaggta gaaactgctc tgttcattga ggagagcttg tggatggcag 9720 ggtgccacgg ctgcgaggaa gaggagggaa gcggacagtg gcacttcctg cggcgttccc 9780 ctctctctga ggagcccctg ttgctgccca tcacctgcag actgtagaca caggtgggcc 9840 ccgccaaaac agggagggac actccacctc caggactgca atggaggacc atgtggggag 9900 cccagaagcc aggcaggagg gcttagttgc tgtgttgcag accctgcatc tgcctgggct 9960 gaggggacag tgggtcccat tcacagtgtc tctggtgata gctgtggcca caagcccagc 10020 ccaggagacc ctgtcaagct tctcactggg cccttggaaa ggagctatat gccagacctt 10080 atgcaaaact cttgacctgt accacctcag ttaaacctca gatcttgctg tctctatttt 10140 agaagtgagg aacctcttgg ccgggtgccg tggctcacgc ctgtaatccc agcactttgg 10200 gaggccgagg caggaggatc ataaggtcag gagatcgaga ccatcctggc taacacagtg 10260 aaaccccgtc tctactgaaa aatacaaaaa aattagccgg gcatggtgat gggcgcctgc 10320 agtcccagct actcgggagg ctgaggcagg agaagggcgt gaacctggga ggcggagctt 10380 gcagtgagcc gagatcatgc cactgcactc cagcctgggc aacagagtaa gactccatct 10440 caaaaaaaag caaaaaaaac aaacaaaaga agtgaggaac ctctttccca agataatgtg 10500 cctggctcac tgtctcacct actttgggtc ctaatcaaat gtcacctcct tactgaggct 10560 ttcttggact gccctactca aatctgcact ccccactttc tctgcttttt ctacgcagca 10620 cttgccgtga catctaacgt gctgttgagt tttcttactg tccatccctc ccccatacac 10680 aacccactag agtgtcagct ccatgagggc agggattttt gtctgttttg ttcgccactg 10740 tcttcctagc atcttgaata ctgtctgtca catagtaggc ctcagtaaat atttcttttt 10800 tttttttgac ttgctctgtc accccaagct ggagtgtagt ggcccaatct tggctcactg 10860 cagcctccac ctcctgggtt ctagtgagca catttggcta aattttgtat ttttagtaga 10920 gatggggttt tgccatgttg gccaggctgg tcttgaactc ctgacctcaa gtgatccacc 10980 caccttggcc tcccaaagta ctggactggg attacaggcg tgacccaccg cgcccagcca 11040 cgataaatat ttcttgaagg aatgaatgaa gctcgggtgg gtttaatagc ttgctggatg 11100 tggcagtgtt gggctcaatc cagggctgtc tgacttcaaa accgatgtgt tgttaattgc 11160 catactccac agcttagaat cagaatgagg atcaaggtat agtcctgggg ttcagagaag 11220 acctgggcct tgccgggaac acagggctca gctccttgga gttaaggctg aactaagagg 11280 ctaacaagga ccctctggat gctgggcagc tcctttgagg agctgggagc ctgagtctgt 11340 gtatcttctc tccactcaaa gtcactggta aagcagagtg cccttatttt tagtgctgtt 11400 gctgttgtgg gactgtaacc attagctagt aagagactta aggaaggaga taaacattaa 11460 tcttctgggc cttccctcag ctgccacctc cgcattgcaa gatgctgttc tcctgcacct 11520 gcccaggcaa ccaagcctga gagttatggg ctggagggtg gtgaggtttg tgcccagaga 11580 gagggccgtg ggtctgtagc tttggggctg gctggcttgg tacctccatc tcaagtccag 11640 ggatggaagg aaggtggggt catgtcaaca tcctgccaga tctggaagaa gcaagccccc 11700 cagccaccag gcaaggctgt tacagcctcc ttgagtgcct cgcttctgga ggtcactggc 11760 cacatccctg tgcctgggac caagggatgc caggtgatct gggagttggg agttacttgg 11820 ggttctcctg gctgcatcct ggtcggtggt catgctgaac ccaggcacag gaaggaaggc 11880 ctgacccaga tctttgggca gctgggacgg attagctggg cagcaggaac taatctctgt 11940 ctgtccccac ctctttccac aaagtagagc tgttgctaga gggaaagttt aggacaaagc 12000 tgggtttggt tagtgaaaca ataaatgtga atttcttcta gtccataatc cctacattat 12060 ctcacactga cagtcctgag tttgaatccc ccttttatcc ctttcctgct gtgggatctt 12120 gggcaagtta cttaacttcc ctgggcctcc gtttcttcca tcatctggaa atgtggacaa 12180 tcatagcatt tacctaatgg gatcattgtg agggctgtgg gaagatttac agaagctttt 12240 tgctgtttag ggtagaggca gggagacagg aatagcttgg cagctatgga tgtgaaggcc 12300 cctgcccggg cctggataat tcagggtgaa ctggactctc ttccttttgc accccctcca 12360 aagcctagag tcttaactca actctcacca ttctttatct ggccataata gcacaggggt 12420 ggagaaagag ggctctaggc tcagaccacc tgcatcactg cctgttcgtg ttaccttagg 12480 cagattactc tatcttttta aacctgtttc ctcggtaata taatagagct aatcagatcc 12540 ctacttcaca gagtttctgt aggtatgaaa tatggtaatc catgcctctg cctgacatgt 12600 agtcagtgca tagtaagcga ttgttatggc gactactgtt attagtaaac ccttattaag 12660 cccctgttta cagaaagaac tctagaaagc actacctgga aaggtacccc cgccttcgaa 12720 gagcttgcaa ctgaaagata actgatgtaa tatatgatgt gagaatcgtg agaagtgcat 12780 tgggaaatcg gggggggggg ggtggagtag gagggagaag tcacagtcta ccgagaggag 12840 cagggaagac ttcatgaagg aggtgacttt tggcaggatt tcagcaagta gaaagaggga 12900 aggacagtgg gggagggctg tgaggcctcc gtgctgtgag tagcatcctc tcttcccacg 12960 tactggagct ctgccttcct gtggaaggaa ttgacccacg cagctcactt ggatctgggg 13020 acttgtggat ttctggttat tccaccaaaa ccaagtaatc ctggagtctg aatttgaaga 13080 ggtcaaagct tacagccatg gtggccaaga ggactccggg gagaagcagg atttgtgtcc 13140 tggtttctct ttctataaaa tgggcatcat actaatgcca cctcctagat tgttatgagg 13200 ataaattaaa agaggcagct gcctggtgta gaagtaagct ctcaataaat gttagctatt 13260 attattttaa gtcatcatta tcttgatcat caacctcttt attatcagca tcattatgtt 13320 tcaggcttgc catcaggact atgtagagaa tatatgcaaa acccctagcc agtgccgagt 13380 atatattagg tgctcagtat aacttagcta ttattagtgt tcctaacaag aaagagattc 13440 tgggccaggc gcggtggctc acgcctataa tcccagcatt ttgggaggcc gaggcgggtg 13500 gatcacctga ggtcaggagt tcgagaccaa cctggccaac gtggtgaaac cccgtctcta 13560 ctaaaaatac aaaaattagc caggcgtggt ggtgtgtgcc tgtaatccca gctactcggg 13620 aggctgaggc aggagaattg cttgaaccca ggaggcgaag gttgcagtga gctgagatca 13680 caccactgca ccccagcctg ggcaacagaa cgagactccg tctcagaaag aaaaaaagag 13740 attctggaca ccctggacca ctgaaaccct gttgtggtgg aaagagcacc agagttttag 13800 ttgaatacct ggattcaaat cccagctctg ctgctcactg gctcgaagtg tgcaaaccct 13860 caagtcattt cctcatctgg aaaaggtggt cataactatc tatctggccc aggcctggtg 13920 gctggtgcct atagttccag ctattcagga ggctgaggtg ggaggattgc ttgagcccag 13980 gagtttgagg ctgcgatcat gccactgcac tcctgcctga gggacaaagt gagaccctaa 14040 aatgaaagga aaacaagttg tctccaggat tgccatgact tgctgcatta cttcagcaga 14100 tcatcacaaa tgcatagtta gtacctgaac tgaaggaata tgaataacaa ggtgaccaca 14160 aggagaatgg atggttgatg gcttttgttt tttctcttct gcttttagat caccagaaac 14220 tagaacgtga ggctcggata tgtcgacttc tgaaacatcc aaacatcggt gagtgcctgg 14280 gcatggagca ttttgtgggt attttgtaga agcagggata acagatatcc actgcttttg 14340 tgtgtgggat cacctctgtc tgtggacctt cacctggtgt ctgtttttac atgagcagga 14400 tagcaactgt gtctcagaat tctggggcat tctagtttag agacctgagt atctgcatca 14460 ctgcggcacc ttctcagggc tggggtgtga ggcatcagaa taggtttcag atgctatttc 14520 ttccctttct ccttctgtct ttgggctgag gtccagggtc ctcagcgtgt gaggttccgg 14580 gctcctagcc tgccagcgtc cctcaccagg ggccatccac agccctcatg caagggtcag 14640 gattttgttt gtggacctga aagagttttg ttcctgctgc ggtgtcctgc acactctggg 14700 ggtttccatg gtgctcccat ttgtattccc cagagccagg aaagcaagct gcccccctgc 14760 ctggctcctc tggcagaagg gatggcagga accactcagt atggggaagg agaaaaaaga 14820 ggatttctcc ctgctcccac cctgactggg gggacaagag cacattgttg gttgtgctaa 14880 agcctgagga ggtttgcctg cctcaaccca ctctggctca gttttacttt gttcagctga 14940 aatggtcttt gccaaaagcg ttggccctga tttggtgctc cttgcagaag ggacagaaac 15000 tgggctggct gcagtgtctg agcagaagcc ccagtgttga cttgaggcag agcaaggagc 15060 atctcctagg ttttccctga aagccctgag tcatcacaaa agacaacacg tgttctgtgc 15120 tcctcaggca tggcctaaat ctcagggctc ccaccgtgcc ccagaggtcg cctgctctgc 15180 tctgttggcg gccagggctg tgaggtgact tgctgaagcc taatgcttcc ttcagagcta 15240 cccagcccct ggcttcccag gtctcgggct agaacagtca aagtgagctc tgtcatggaa 15300 gggctgaggt cctgctctag ccctctggga gaggagcagc tctgaggtag tcagaacgtc 15360 agctgtgcag ggctttctag atggcaatca gcagcttgga ttacacccga agcagattgg 15420 tgtggccagt ggtgatcggc tttgcctgat gcagtgtgtt ctgcagagcc agcacctctc 15480 agctggtggg ttcctggccg cagaactact ggagctccta ggtggtttct gaggttaggc 15540 cttcacctga aaacagcgca gtggggactg acatgttgcc tttggtagga gagggcccac 15600 agagggaaac acctagaaca gcagtcacag attaggcatg ttttgcttgg ctgactcagt 15660 ggtctaaaaa tatttttatt atttgccaat atttaaaaat gagatttcac attttgaaaa 15720 aagaaaaaat ctattccccc gcctttccag tcagaaggct tggctctgct gagcccccac 15780 cttgcatggc cagaaggagc tgtgaggagc ggtggctgcc cctgcagccc gctggccact 15840 gtccttgtca cccactatga gctcacattt gcattaccca cctgggcccc tgtaggcctt 15900 gcaagcttgt gacctctaac ctagaagttc cagaacagga agaaaaaaca tgtgcgtgac 15960 taaagccacc cataagcaca gaagcatttt gatgttccag acccgggtct caatatctga 16020 ggagggtaac ttcctttcct ttatgctcct tgtgaccaac tggtacagca gtgataattt 16080 gtcctcatgt aggcaggaga acagcagcta ggggtcagtg atgcaggaag cagaaccatg 16140 tccacatcac ccgcgatgcg ggcgggttga ccatgggcgg gttgaccacg gatgggttgg 16200 ccacggacgg gtcagggtat aatgaagaca attgagaaat gagcaggaag gacaaaaata 16260 gaattctagg tgaaaaaagc cctaggtgtc tttttattta tttctagaat taaatacata 16320 cttttttacc ccatagactt cactctgttt ggtagccctt tacttttacc atctgccctc 16380 ggctcagaat ggaggcaggc ggagggacca tatatcctgg ccgtctgctc agaggccagg 16440 tggggcacag tcactctttt ggcctctgat ttcctagaac tgtgcttcca tttcatgact 16500 gctcccaggt cctaaggagg ttggtccgag gaccgattct ggggttgagg gtgggcagag 16560 ggaaggggga gtcaagactg tgtcctggga gctccagcat ccggtgggaa ccagggctgt 16620 tggagatgtg gcggagctgc aggtccaggc ggctgtggtt gccatggatc tggacctggc 16680 ttgtggcagg agaggaggca attttgtgcc cctaattcac tattcctctt ctctctccac 16740 tgcgctgtcc ttcagaactg tgaccctttt ggctctggcc tcttgaactc catcccaaag 16800 ggaaacaaac gggccagccc aagaacagtg cacagtcgag gaagctagag caaagagcat 16860 gtggtcagcc ctgcctgtgg tcagactcgg aggcactgaa ttcagatgga gcatttggtg 16920 ctaggggcca gtcatgccca gtttcccctt aatagctagt atattctgtc ccaggagtta 16980 aaagcctgtt ggaagagtga accctgatat aaactctgga ctttgggtaa tgatgatgag 17040 tcaatgtggg ttcatagacg gtaacaaatc caccactcta gtgggagatg ttgatggtgg 17100 aggagactgt gcatgtgggg gacgtggggt atttgggaat gttctcgggt atttgggaac 17160 accctgtact ttccgctcaa ttttggtgtg aacctaaaac tgctctgaaa ataaagttta 17220 ttaattaaaa acaaacaaac aaacaacaaa atgcctgttt gggtgtaagg cacactgccg 17280 aactccaaac agcgctggga gtgtggccag tggtggggag ttgagaggag gagacgctgg 17340 tgtgaggtct gaggtctgaa tgaagtccgt tctacctgtg atctgcctgc tccctgctct 17400 caagtcctct aatgaataga ctctgtcttc cttcgtgctg agctgcccca gcagtttcga 17460 tcatagtcta gcattgtggt ttagagcagc acttctcaaa cttttatgtg cttaagactc 17520 acgcagggat catgttaaaa ttcagattct gattcagggg gtctggggta ggacctgagt 17580 ctccagctga tgctcatgct actggtccgc atgcctgtca atacttggag aagccaagtt 17640 ttgcggcttc ggagtcgcat ccagatttgg ggtttgaatc tgggatttgc taattagtaa 17700 ctgtgacctc tggcaagtta tttaactcct ctatgcctgc ctctgttttg ttatctgggt 17760 cccttcgtgg agttgttatg aaagggttca gccaggaaag ggggctagga gggagatgat 17820 gaaaatggag attccagccc ctagaagtga tctcttcaag acccccagcc tcgactcagt 17880 tcacaagtta ttcaagcctg accatttacc cttgagccca gtacccattc agctaacagt 17940 aagtgtagca aagaaacggt tgcaaataaa aagaaacatt gaatcatgac tgagcagttc 18000 ctacatccct gcccccatgg tgggggtggg gggagccctg ccacagtaag ctcttggggg 18060 gcagctcagt cccccacaag cccccatggc aacaggacct ccttcccact gtgttattgc 18120 tgcagatatt tttaacagca acactttttc agtgcttttt ggagaaagat ttgttagtta 18180 aaatgtggca tattgttggg tggtttttaa agaattggaa atagccacaa catttgggtt 18240 gtggctatct cagtccttga agacatgaaa tatcaagtaa aggtttgtag gtgttttggc 18300 ctgttctgtc ttccacggtt tttaaagaac agcaattagg tttgttgctg aaatgcagta 18360 aatgctttat actcctttcc ccagatcttc ctgtctatgg acatggcctg gcccttgttg 18420 gccttcatgc cctgtcttta ctctggaatg ggctgggtgt cagattattt tattccacgc 18480 atccatagtc cctctgctcc tgcctcacag catgacacag ttgtgcttag ttaacgcatt 18540 tgtgtaattg ctggtttaaa gcctgtcttc cctcttcgcc tggcagctcc aggtggcagg 18600 gccggctcct cttcttcaca gccacatcca tggcatgtac agcctcgcct gctccggggt 18660 agctgcccag tggacattgt cgagccagtc agaatggcca caggtagtgg ggacagattg 18720 gagctccttt gcctaagaat ttgagaaggt gactcccaag caactctgca atatcaggaa 18780 tcttgatgtt ggtttgtctt ggcttcaagt cccggttctg ccacttagtg tgattttggg 18840 caggtttctt atggagcctc agtttcctct cctgtcagat ggggttattt atatgtaagt 18900 agctaccctg cagagctggt gtgagggttc aatacagtaa tgcacgtgga gcccatggaa 18960 cgatgccggc acacggacag ctcaactaag tgttagttgt tagatttaga ttgttattat 19020 cagaatctga tggggtgcgg tggctcacag ctgtggtccc agcctctcag gaggctgaga 19080 caggagatgg ctcaagacca ggatctccag cccagcctgg gcaacatagt gagaccctgt 19140 ctcttaaaaa aaaaaagaaa taatgaatct gctgttgcta aataggcact tagaatggca 19200 cagtcatttc tcctcttgtc ttcagtgtcc tgttaatttc tttacaaatt aaaaaaatgt 19260 cgatagcagt cttattcaga tacagcttcc tccatccctc cttgtcttgg caggtgcctt 19320 gctctggggc acacatcaaa gctgttctct ctgctgggtg gcctagaagg attagtcttc 19380 ctttgctgct cctttcttct aattcccttc cccggcttcc tcccacctgg gctctgtgtg 19440 tggccttcct ggagaagggc agacgccaat gactccatgt ctaggcagag gcctgggtgc 19500 ctgcacttct tgccctgttc ttggccttgc tgtgctgggc gggggcaggg tggtgtgggg 19560 catggggtgg tgttgggcat ggggtggggt tctggctgag gcagggctca gtgccaggcc 19620 caggcagagc tgagtggctc cacttctctg agatggttgt cagcatcata cctgctgctg 19680 tcccgttaat tccccatgct gctgctgtta gtcacctccc taatggagct ggtctgtagc 19740 ttctgggaca gctgatttcc aggggattat ttgtattaca cactttaatg ctttttaata 19800 gcaaattttt aattaaatgg aaagtccttt tggaagcgag ggagcagcag ctgcagcaag 19860 actcagcgtg aggcaccgac ttagaccaga ggtgcgcaag tgagtggggc ggaggcaatg 19920 gcaggacttc gagaggactt gattgagtgt atatggagtg tgcccaggct aatttttatg 19980 ggaggaaggc aggggcctgg cgctggctcc ttcctcctgt cctaaaagcc ccctctgtca 20040 tctgcaggcc tagggaagca tcttctttgc ccaggagaga atgtatattg gatatataca 20100 ttatatccaa taatgggagg gatattggaa gtatcacctg cctttgatcc cgttcccaga 20160 aatactgaga ttgggatggg atttttgggg ttgagtcact agattagatc aaatagtgta 20220 ggtaatggga tgcggaaaca gtcttgaggc cctggctccg gccctggcag gcttcggagt 20280 cctcagtcat caagggagga gaacaagggg gctatagtgg tggttcagtg cctcgggact 20340 gtgccggctg ggttgtatac tttgctttct gaatgatctt gcttcgtggg gaggggacat 20400 agggaagcac ctcagccctg aggaaacgtg tgacactgga aatggaagca gccagggccc 20460 acccaggaag agacatggcc atttctttgt ctcctagcac tgaactggtt agtttggtgt 20520 caggccattc ctgaagtgct ccatgaggtg cacctgtaac tgccaaggct tggagcaaag 20580 gtcaaaccga gggaggcctt tggaacagaa gttccccatc aagagagttc acgtgagggg 20640 agggacagga cagtcagcca aagcggagtc gtttctgcat tagaatgatg ctcaggggtt 20700 ggcatttaac ccagaggtgg ctttgtgggc agaaacttga agaggagacc tcagaagact 20760 tcaggttggt tttttaccca agagctttgg aggcggggag cagggaggga ttccgcctgc 20820 cagctttttc tcgcagctgg tgcatcgccc gagtcttctt ccagtggcac cctcccggac 20880 ctgtctgcga tgctgcttta gggacatttg taagtggtct ttcttttgga tgccagggct 20940 ttgttgcctg aatatggggg ctgccccaca tttcttaagg gaagcagtgg tgtagaccac 21000 agtctttgga gtcaggtagc actggattca catcttgacc caccacttag aagctctttg 21060 gcctttgtta agagactttg tgtccctgag cctctggtgc cctcatctgt agaatgggaa 21120 taacattcat ctcaggtggt cgaaaggaat aataaactcc tcaaaggcag gcactctgtc 21180 tgttcctcct gaatcccgct gcctagcgtg gggtccagca catagtaggt gcttgataaa 21240 tgcttgcaga atcagtaatg tatgcaagag cctagcacaa ggcctggcat agtaagcact 21300 taataagctg ttattgttgt cattgcctga atgtgtgcgt ggccttccag gctcaccatc 21360 cattatcctg caccacgtgc cttcctgctg agctctgcct ttccaccttc ttccccaccc 21420 cttagttctg ctcccattta ctgctctgga agagctctct ggctttccca tctggtcatt 21480 gttgtcccct gccgtcaaca ttgctaggtg ctgctcacgc tgcatctcac catcgtgcat 21540 catatcccag gaccaccttc tcggagacca gccctctggg aaggttccgg cttttctcca 21600 tcttgacttc ttagccatga agcttttctc tcttgcctga gtctgaggtg gcaaccagag 21660 cgccaggctc tggctcccag gctgcatagc cttgcactgg ggggcactgg gcacgtcgcc 21720 acttcccccc actgctcctt ctggagagcc ctgtgagccc gacaggatgg ggcaggggtg 21780 gggctgctga ggagaagcct aggatttcca agttttctct ctgttaatct ctgtccccat 21840 ctcctctctt gcagtgcgcc tccatgacag tatttctgaa gaagggtttc actacctcgt 21900 gtttgacctg taagtgccac tttctgaggg tgtgggggcc tttccctcta gctgactcaa 21960 aatgaaggct caggaagggg cctaaacagg ctctccagcc tccgcccagg gccccctcct 22020 ttgtccgagg gaaaggattt gactggggca gattgctgcc cccaccaagg gggtctccat 22080 gttcccccag cgtcccccca gggctctgaa ccccaggaca gcattcctct cgcacttctg 22140 ttcagcagca cgccttgcac agatgccttt gtcttgtttc tcagtgtgct gtccttagtg 22200 aagaaataaa agacagctct ttgcatgacc ttaaaaatcc tgagaaatca gaggtagctt 22260 tcattagtcg gaaaccaggc tccattggat tgggtctctc ctccacgttg gttgtggttt 22320 aatgtcttaa aagtggctct tacctcctgg acactcctct ccaggattct cagggttggg 22380 tctctgtgtc attggtctca ttactcttca acttcagtag tagctctgtc cttcctgggc 22440 agcgatattt tagtgtttat gttggtctca aagctgtgac ttttggggta ggttgactgt 22500 tttctcttag atccctgtat cttcatctct gcctgactat tagtgaatct gtgcattttg 22560 gaaaaagaaa tgtccggaag gaagggacgg cccatgatac ctcaaggaga atccgggtgt 22620 cactgaagga tcgagtgtgt tctgagctct cagatgaaat gcatggggag ttgggatttc 22680 tctgaaagcc attctacagg gtgaccctgt ttcttcttgg acattggggt tggacaaagg 22740 accctttctg cctctgaccc tcttcttccc gttggttgca gtgttaccgg cggggagctg 22800 tttgaagaca ttgtggccag agagtactac agtgaagcag atgccaggta ggatgagggc 22860 ccgagagttc aaatgtagct ctggagttta ggactgaagg aagtcttggc caccttcggg 22920 gtccagcatt gtacctgttt gaatagtctt tggggaagat cagaatagct cttgtctgga 22980 gaaagattct gttgagctgg gctagggctt gcatactgtg ggtgatatta gaagttaaaa 23040 attcagcact tcctaaccag gcgcagtggc tcatgcctgt aatcccagca ctttgggagg 23100 ctgaggcagt tggatcacct gaggtcagaa gttggagccc agcctggcca acatagtgaa 23160 accctgtctc tactaaaaat acaaaaaaat tagccgggtg tggtggtgtg tgcctgtaat 23220 cccagctact taggcggctg aggcaggaga atcacttaaa cctgtaagcg aaggttgcag 23280 tgagccaaga tcatgccact gcactccagc ctgcgtaaca gagcgagact atgtccccct 23340 cccccccccc cacaaaaaaa atcacttcca aatgaatgtt ttacaaagct tttccaagtc 23400 tcctttaccc tgtgacccca gaaatacttt ttttttgcac taccatgtac tcgccaccat 23460 gcccaatgtc cccctctgcc cttttctttc ctttgacaaa ttctggtgtg ctcaagccac 23520 tgtgctgagg ctctggcatg atccagaggt gcagaagaca tggtttctgt cctgagggag 23580 tggagagttc tgggctgata atccaaccat agagccccgg gagctttcag cctctgtcac 23640 cttgtcccta gaccaccatg accagccttg ccgtggggct cctccaactt gaggaccgtt 23700 ccccggccac atgcctcagc ctctgccctc cctggaatcc ctggtgcctc cctcacccac 23760 gctctcaggt gcctgttcag cctgcctttc ccgccttggc tcttccccca gccttgcttt 23820 tctcgagggt gatgtcccta caacctggtt ttgatcatcc tgcctgcagc ttatctggct 23880 tatgtggcag ctctggctgc ttctggagag tgggggagtg cagcttcctc acgaatttct 23940 caaccttgag aggccaatgt ttgctgatca acttcagatg cttcagcctc gggaagaatt 24000 ctcaagtggg gagatgaatt ccagtgccag caggggagga cgaggctctg ggacggagga 24060 ggcagtgatg gctcagggag cctgcgggga ggagggagag ctatagggag ggggccctga 24120 gggggggtga ctgtaccagt gggcttggcc tggctctgct gggacacttc gcacttttgc 24180 catttttggc cagaaggcgc tccctgctag cccggctctg ttctaattat acatctctgt 24240 ggagactcgc ctctatagct cagtcttaaa gtttctgtgg cccactcttg ggctgtgtcc 24300 tatggggagg cccgagtttc agcccccagg gacccagtac gaccccttgg ttcttgtggc 24360 atccccagca tcagatttta ggaatagtaa gtccaggcca cccagcccca tacactggga 24420 tgctctgcag atgtgcttaa tataccagat agtgcctgat gacgggggtc tatattctag 24480 gccaagttcc tcagccttgg tgctactaac gttttaggcc aggtacttcc ttgttgtgag 24540 gcctctcctg tgcattgtgg cagacattta gaagcatcct tggcctctgc ccaccaaatg 24600 ctgggagcac ccctcctcca gttgtgacaa ccagaaattt ctctaggcat tgccaaatgt 24660 cccctgcggt gggggggggc gggcggcaaa ttcattccca gttgaaaacc actgctctag 24720 actgcccccg ctccctgtca ggagtttgat gacagggatg gcaggatggt ttgctatgtg 24780 gacagtctga tttacgtgtg tgactgtggc tgggcgcagt ggctcacgcc tgtaatccca 24840 acactgagag gccaaggtgg gtggatcact tgagctcagg agttcaagac cagcctgggc 24900 aacatggtga gaccttgtct ccacaaaaaa atacaaaaat tagctgggca cggtggctca 24960 tgcttgtggt cccagctact ggggaggctg aagtgggagg attgcttgag cccaggaggt 25020 caaggctgcg gtgagctgtg ttcacgacat tgcactctaa cccaggcaac agagtgagac 25080 cctgtctcaa aaataaaata ataaaataac tttgggtttt tttctctacg caaaatcatc 25140 agaagtgctc cttaaatgcc ctgtttggaa gctcttaagt acattgtttc ttaaaggtat 25200 ctttgtactt gttttagctg ccttactgga tgccagacct cagggcagct attgggtctt 25260 gtccatcttc attatcctag gcactcaata aacatttagg gaaatgaatg agtgcaccca 25320 ccgccaaagt agcttaggtt gtttagttgg actctccttc ctaagttgcc agcacaagct 25380 tcttctccaa gaacaaagtt actgtatgga gaaagagaaa gaaggaaggg attggatgct 25440 ctcttcttcc tcaggattct gggctgtctc ctgatctctt ggaaatgagt tggttgtgtt 25500 agacctttcc agtcaaaagg gggtgagagg aacccgttct agcggtgatc ctagaaaaac 25560 cattgcatct gcctgggcct cggtttcctc tttctttaaa taggttgaac aagatgatgt 25620 gcagagtcta aggttccagt ggccgttaag tgattctctg tgaatccgtg gccccttgtc 25680 acatgcctta gtctgcagca tgtggttgtg gatgtggatg aggtggttta accctgcgct 25740 aacatttctt ttccttctgc ttttttagcc actgtataca tcagattctg gagagtgtta 25800 accacatcca ccagcatgac atcgtccaca gggacctgaa ggtactaccc aggctcccct 25860 ccgtgcctct gctcatgaag tgttggcgcc acctggtgcc agatagtggt actgcgtagg 25920 cccaaactag gcttcctctg ggctgcaggg tgggtgctca caaggttctc tgtgtttctt 25980 ctgcagcctg agaacctgct gctggcgagt aaatgcaagg gtgccgccgt caagctggct 26040 gattttggcc tagccatcga agtacaggga gagcagcagg cttggtttgg taagggtgat 26100 cctgtcttcc cggaatgcag cccccgccct tcctcctctt cctgatctgc cttcctctat 26160 tagaactaga agccagaccc ttaatggtcc tggcctccga gatctctctt ggccgtacgc 26220 gactcagtac agtaagtcta gctgttgtca gcactgcttt cttgctgcct gtgggaagga 26280 gctggagttc ctggtaggca tacggctttg ccgtctggtt cagattccag gcgctacaag 26340 aagcccagcc tgtcagctct tgctgcccat gtgctgagag tttatgtagc aaaagcagca 26400 ggaataagat gggacttggg ggaaatggct ggtgtggatt taacgagaga gaaagtgggt 26460 tcagtatgcc tctgccctct cttttgctac aggttttgct ggcaccccag gttacttgtc 26520 ccctgaggtc ttgaggaaag atccctatgg aaaacctgtg gatatctggg cctgcggtaa 26580 gcccattcca cgctctcagc ttttcgctgt taagggccct caacttccga tgatggcaag 26640 aaagaggcat cgctattcct tgcaggtcac acacgtgcct ggtgtatgtg aaattatggt 26700 gtttgcccct gggatggctg ttcccatcac accctcctcc ctgcgtactt ctgggatgac 26760 attgtatcct tcttggagag ggatttgccc acgccttaga ggatgggttg tgcctaaaga 26820 aatccctggt gtgacttggt gacgtgaagt gtgaggcata gcaggagggg ctggtagcat 26880 agcattatcg gctggcatcc acttctgact ctggtatggc ccctgccttt ctaggtggct 26940 ctgagccctg catggttttt cttggttcct cagggaagta ggcgactgac ccccatgacc 27000 tgtgtgttct gtctcgtagg ggtcatcctg tatatcctcc tggtgggcta tcctcccttc 27060 tgggatgagg atcagcacaa gctgtatcag cagatcaagg ctggagccta tgatgtaagg 27120 accagagagc cgggcagcca ggccaggaag ggcagatgtc ctgctcctcg ggctctgtcc 27180 aagggagcag gcttgtttag tgtgtcacgt gatacggggg tgtcagggga ctttgaggac 27240 ccaggaatgg gcatccaggg cccaattctt gccactctat gtcccaggga gcaactttct 27300 tttgcacagc cttcttcata actaaaattg aggagtccac tgaagtcctt tgatctttac 27360 ttgcaaagaa tggagcggcc tcattggtgt gctgtgtaac acagggacaa aaggcctgga 27420 gactccctcc actgcagtgg caccttggac acattgctga gcctctgttc cctcctaagt 27480 atagagctgg gcttaaacca gagaatgttg gagtcccctt cccgctctaa tctgatgttc 27540 tggcattcta aacatgactg ttctgtctgt ctttccaagt ctttaagttg acacaggttc 27600 tggaatagcc gcagggcttc tccaactctg ccagtcacag ctttaggtac cacagagtat 27660 cccaattaca ggagttgagt tgaagacaga accagtgttg cagggtatga agctcaccaa 27720 taccacattc ttctccctat tcctgctcct tagttcccat caccagaatg ggacacggta 27780 actcctgaag ccaagaactt gatcaaccag atgctgacca taaacccagc aaagcgcatc 27840 acggctgacc aggctctcaa gcacccgtgg gtctgtgtaa gtgtctttgc tagtggccaa 27900 ggagctcagg ggtgtcagcc ttctgtgtgc cctcggcacc accccctcct tcttaccagc 27960 agagattcat tctgggcccc aagcaataac tgagcaggcg ggcagaggac tgttgagggc 28020 cagggtcaat aaatgtcacc agggagactc gggaggctga tggggctggt gggccactgc 28080 tcctctctcc cccactcatg gctgtcaggc tgggattggt tctgttcttg gatgagggct 28140 caggttgacc cttgtggact ccaggtagcc ggtgatagaa agcagctggc aaaacccaaa 28200 gtgaattccc aagctggggt tcatactcag atctcaactc cactggagtg gtgaccaaga 28260 tccaacaaat caacagaagg ggtttctgag tcattaaaag cataaaagct gaggcataaa 28320 gcttctgcgc taaagtccta ggagagtcct ctaggctatc agtgtgggtt gacgtactct 28380 gtttttatac acaattcttt caagctgaaa tatcaacttt cagacaaaga agaggatttg 28440 gtagagttag gcatcttgac aaccacgagg cattatttat ctgtccattc tgtgtttatt 28500 aaatacctct ttggtgctgg ttaccgtctg ggtgctggag atacaaagat gaatgaggca 28560 tggtccctgc cccaaaagat catctaggga gacaggcact caaacaggca gtcatgttac 28620 aatgtgacaa gtaggtacaa gaatctaatg agagtacagg agctcctact gttcctggtg 28680 ggtggtgggg ttactgaagg ctgcacggag gaggtgacac ccctgtgctt gttcttggca 28740 aataacgagg tcctcagaac gttaacctgc agacagagtt tagcacagtg agaggttatg 28800 ggaaactatg gtgagttgaa ggaatgttga gttgtttggt tgtcgatgag gctgcaaata 28860 tcagaatgca agagaatggg gcaaaagatt ccctgacata caagtttctg cctcaggagt 28920 ttggatttta ttctgaaaac atagggaatc atttaagggt tttaaagaag aatgaaattt 28980 gcatttaaga acactttgga agttgtgagg aaatgaattg ccaggcatgg tggcatgtgc 29040 ctgtagtctc agctgctggg gatgctgagg caggaggatc ataagcccag gagtttgagg 29100 ctgcagcgag ctatgattgc acctgtgaat agtcattgta ctccagcctg ggaaagatgg 29160 tcagacccca cctctttaaa aaaaaaaaaa aaaaaagaag ggaattgaaa atttttaaaa 29220 gaaaagggct ggagacagag agctcaggaa gcttttttaa tagttggaat agtctaagca 29280 agaccaggtg aggtctcagc agagggtaag gatgggggaa tgtgcagtgt gttgaaattc 29340 aagagatatt tgagagaacc taaaggattt aattctctcc agttggattt ggggggagca 29400 aagaagagag aggccaggtt tcaagttgag cggagagttg taccctcact gacccagaag 29460 aaaaccagag gaggagcttg tttgtgagac aagacgatgg ttttctcttt tttttttttt 29520 ttttgagatg gagtctcgct ctgtcgccca ggctggagtg cagtggcgcg gtctcactgc 29580 aagctctgcc tcccgggttc atgccattct cctgcctcag cctcccgagt agctgggact 29640 acaggtgccc gccaccacgc ccggctaatt ttttgtattt ttagtagaga tggggtttca 29700 ccgtgttagt caggatggtt tcgatctcct gatctcatga tccacccgcc tcggcttccc 29760 aaagtgctga gattacaggc atgagccact gcgcccggcc aagatgatgg ttttcatttt 29820 gtgcctgctg agtctggcaa cctccagcca gacacattca gtgggtggtt agaaatatgg 29880 tcctagagat tagaaaagaa gctaaaaatt ggaaatccac attgtagtca tttctgtgta 29940 gttggtagtg aggctgtaga aatagcctct tcctatgctg tagatgggcc tgttcctatg 30000 ctggttgagt tcttacggtg agcttctatt ggctgtagta gagaagagac ggccactaca 30060 caccagcatt taatgatagg gagagttagg gggcccagca aagagcactg agagtgagac 30120 cttccagaag acccagaagc taagaaacag ggggtctcag taagggagcg tcaggaatca 30180 gatgcagaag agtccctgat taagttgggg aagaatcccc tggctctgac cattagatgc 30240 cattgtttca tcatttcact gagacagtgg agagaaagat gaaaccctgt tttcagtgag 30300 acgaaaaggg agtgagggtg aggaggggca tggggagcta ggcattgagg tgggaaataa 30360 atggtgatac ttagattaag atgggccagg ggagctttta atgtaaggct cacacctgta 30420 atcccagcac tttgggagac caaggcaggc gatcacttga ggccacgagt tcaagaccag 30480 cctggccaac atagtgaaac tccatctcta ctaaaaatac aaaaaattag ctgggtatgt 30540 tggtacacac ctataatccc agctacttgg gaggctgagg catgagaatc actagaaccc 30600 aggaggtgga ggttgcagtg agccaagata atgacactgc attccagcct gggtgacaga 30660 gggagactct gcctctaaag aagaaaaaat ttctttttaa agattatatt ggtcaggagc 30720 ggtggctcac acctgtagtc ccagcacttt gggagaccag ggtaggtaga tcacttgagc 30780 ccggaagttt gagaccagcc tgggcaacat ggcaaaaccc catctctaca aaaaaaaaaa 30840 ctttaaaaat tagctggttg tggtaacgtg ccttagctac ttgggaggct gagatgagag 30900 gatcacctga gcctagagag gtggaggttg cagtaagcca ttattgtgct actgcactcc 30960 agcctgggca acagagtgag atgctgtttc aaaaaaaaaa aaaaattttt ttgtttaagg 31020 agaggcttaa ctataatcta tagagaagaa tctagtccag aggaaagagt tgaagatcct 31080 tgctaattga ggaagcaaag gtttggacag cagaaaaaga gagggggctc ctgagccaag 31140 ggcagggggt ccatcccggg gatgaccatg atccccctga gacttctatt agtgtggagg 31200 caggtgaaga tcggcttgtg agtggaagtc tgagctgaaa ggggttcttg ctgatgacct 31260 ctcattttgc ttttggagaa atttacaccg aggaggaggt aaaatgagag acttggggaa 31320 ggtagagaag gtggggagag ttgcctccgg acctggaaag agtgggccaa gggtgaggga 31380 aaggatgcga ggaggccccg tagtgttggt gggcacctgg ctgcaggtgc caggatttgt 31440 ttttctgaca ggctggtgaa gacagcaaca gcaaggggag agggcaagca acctgaaaca 31500 ggcacccaag aatgggggaa atattctgtt ctggggtcat ttttgcaggc cctaccctct 31560 gcagtcccgt gtgtctcgag cccctgagga catcactata ttctgaaatt acataatgat 31620 gctggtattg acagctgagt cattgaggaa gtgtagactg tgtcccatgg actctgttta 31680 aggaggccag gaagttagca gtaaatacat tgaagacaaa tttccatcca aaaaaggcgg 31740 ggcacagtgg ctcacacctg ttatcccagg actctgggag gccgaagtga gcagatcact 31800 tgaggtcagg agttcgagac caccagcctg gccaacatgg ccaaccctgt ctctactaaa 31860 aatacaaaaa ttagctgggt gtggtgggat gtgcctgtag tcccagctac tcgggaggcc 31920 aagacaggag aacctgagag gcggaggcta cggtgagccg agattgcacc actgcactcc 31980 agcctgactg acaaagcgag actccattgc aaaaaaaaaa aaaaattaca tccagaatga 32040 tgaaaagaat tgatgcttca aggtgacgat ccttagcttc tgggatcatg gcttcattca 32100 ggaccttgct gggggtgtgt ggagaggggc tcttggaagg aaggaatgtc ctctgtagag 32160 agcaggaacc ctgccgttct ctcgctgctg agcatctgga acgcagtagg tgctcagtaa 32220 acagctgcct aaggagtgac tgaatgagga tcacagcccc cagggtactc tcctgttcgg 32280 tagcctctgt ttcccaagga agaataggac ggtctctcag cagcccgtct agcatccgtt 32340 atggtgttct cacgttcatg ttgtccttat gtaaccttga gtttcgggta gtgcttttat 32400 tctaaaagcg ttttcacatc tgtgacctca tttcatcttc agagcaactc tggggtggct 32460 gagtgcatga ccctgtcctg ggcatggtat cggtgccagg actgtgggag gcgcagagga 32520 tctgggctgg ggctcatagc ctgtctgttt ggtttctagc aacgatccac ggtggcatcc 32580 atgatgcatc gtcaggagac tgtggagtgt ttgcgcaagt tcaatgcccg gagaaaactg 32640 aaggtgagtg tcgtttctag gctgccagcc tccttgacat catgccttgc accagtgtgg 32700 ctcctgcccc atttcagaag gaagctcccc tcctggctgg agctgggctc tgaaggttgt 32760 acatgtcaca ggggaggggg cccagaggcc tgatgtcttc aggctctagc caggacctgc 32820 ctttgcctga gaccagcctg cccttttcta gggtctcagt gaattcacag gaccttcctc 32880 tttccccagg gtgccatcct cacgaccatg cttgtctcca ggaacttctc aggtatgttt 32940 tcccagctgt gtactttgat tatgccgagg tgagtggatc aggaatgggc tgttgccatc 33000 ccgggcaccg ctgggtttcc tcggcgtcct gggccacacc ttgaccaggg cgagtgagga 33060 tcctgttttg aggggctgct gctgctgctg agtcctgctc ctgagattca gggggctgga 33120 ctcacatttg tgaattgttt cctagaactt cccaaggagt agcctgccca acttgctatg 33180 taccttgttt ctctggattc ttatttaact ctctgaagac tctcagcact ttacagattt 33240 tagccattct aggatcttgg aggatgtgct gggggaagaa aagagagatg aggtacagtg 33300 agtcttctca attgccaaat tgccaccatt catttgcctg ctgggacgat ctcttacttc 33360 attttgtcca agtggagatg actaatagaa attattccag atgtttaaac cttttgtggc 33420 gacttgtgct taaaatagtc cctgagatac tagctataac agtgaagaaa taaagaccag 33480 caggagagag ggaaaggaac ttgcttaaat ttgcataaag aattgggaga ggtgggacca 33540 ataatttgta aatcatactt gacatttatt tttaagatgc aagacactcc actcccctct 33600 tgcccccacc ctcaccccaa cccctattat tgtttgcctt caattgggaa gcacagtggc 33660 ttttttgtga ggaaaagatt aatgtcgaga ctgaagacag agagggctct gcccagcttg 33720 ccatctcccc cggtcctccc tccctctaac cccttgcctc actgttttgg ttcaagaccc 33780 ccccttctcc ttcccataat aagactccct cccttgcttc ccctctgcac caccatggaa 33840 agggggttgt gtgggagcct aagccaccac tcagtgggag ccacttctga atacccgtcc 33900 tgctgggctc gcctgcgctg gctccaggta acgccagggc cttggctgtg aggatgctgc 33960 aggcagggag cctagggctt cgtggtgtag cctgagagcc atggagctcc ggaaggccag 34020 ggctggatag tgagcccggg gctggtggtg ccctgcccta ggccttctcc tttgaccctg 34080 gtttggggct tgatcttgtg tcatgggtac ccacgacggg catactgtgg tgtggctcca 34140 cctctcgcag atgggaacag ggaagcgtgg ctggctgcct ccggtggagt tgcaactgta 34200 gtcccacact tgcttcttgt gctttaatga cgcagcttct actttttggg tctacgagcc 34260 tttccagagg acattgaagg gcgtttcggt gttgccccta gagcgaagct ctgtcctctc 34320 tcccctctga gttgaagaaa tgtgaagaca gtctgctgct tctcttttag cccagccagt 34380 caatagcaag ggccctgtct tgcagccccg ggcctccaca tcagcctccc cctccatttc 34440 aggaaactgg catcctggtt tcaggaaatc gggtgttagg acaaagcatt ttattcatcc 34500 ctgtagagcc tcctgttctt attggccaga cctagactgg cctttgagct cactttgcct 34560 tgggtcagag gagacaaaca atgttgcaag cattccagga tggcctcttc tgccctgact 34620 ctgggacagg tgaggacaga gtctgtccgg aagcttctgc agaaagaggt gtctatggat 34680 gcaatcaaga aggaagggca cctgtgtgtt tctctagggc tgttttttga gttgacctcc 34740 aataggagat gtggcttatc ctggactcta gcagtttggc taacagcgaa tcggggcctc 34800 cagagtgtat tgcttcagca gcctttgttt tctttctcag ggtttatttc ttgggcacct 34860 ttcacctcag cacactgtga cacacagact gagaatgctg cctctctcgg ctacctccct 34920 taagacaggg acctgtgtct ctgaggggtt ggggggcatg gagctggggc ccaccagtaa 34980 acttagctgc acaagggcca cagaccctcc ctgggacccc cacgccagtc cctctagtgt 35040 gtgggatgta gagaggggag agggctgctc tgcgcccccg gcactctcat cgtgggctca 35100 tttagcttct agggagggaa ggactagaag ggagggcgtt tcatcacagc cttaagctag 35160 ggccgggcta cctcagaagg ggcacctgcc tctcaccggc tcaggcattt cgctgtggac 35220 cctcctccgg agggggtcat gagacaggca ctgcagccct ctccatctgg tggggacgca 35280 gtgttcccta tgccctggcc cagcccggtc ttcccaggcc cccagactgc tgcagggctg 35340 gctgcgccta cctcctcagc ctgcccctgg cgctccgctc ccccagctcg gctggcttgg 35400 ccacgccgcc tgggctgcgc ctcgcgctgg ggcatgctcg ctgctgacgg ccccgtggct 35460 ttgcggggct ctgtgcactg agagactgta tcccctcagt tggcaggcag agctccgccc 35520 ccgcctcgcc tgccgcgagc gccgccggcc tggccgggca aggtacgtgg catgagtcct 35580 ccccgaccgc ctgcctcggc cccctgccac cccaccagga gggccagcat gccgggccca 35640 ctcaccaggg aggcgagtcc catgcttgcg ggctgagatg ggcatgccag acagactacc 35700 taacttggca tctgcaagcg catcgttgtt atggagcccc ctaaccagcc atgcatgctg 35760 ggcgcttgcc aactttcagg gggcagtagc ctgggggcat ggagctgggc agcgggagcc 35820 ttgccaagag cccgatgccc tgggagggct gcagccaaca gtgggccctc agagacagtg 35880 ctgggcattg ccctgagctg cccggtgcta ggactagatt tccgcagcac tgtttaagac 35940 cccacagagg agccgcgctc ctcaaaattg tgaagtctgg cgcttgctgg cctccaggtc 36000 tgaaaggctc cagagtgcag aagcctcaga gccagctgtt tctgggttca catcctagcc 36060 ctgccacacc ctgagcgagt cacaccagct ctcccagcct taattcctca cctctccaat 36120 ggggatgata aataacatgg tggtgcttaa gatcaccctg tcgaaggctc tcagccctgc 36180 ctgtgcagta cagctgttac ctgggagctc gtaagaagtc ctaatgccag gaccccaccc 36240 cagacaataa aatcagaccc ttagggataa gataggtagt acgctttttt taagctccca 36300 ggtgatccta gtgggcaacc agcgttgaga gctggctggt gaatggaaag cacttagaca 36360 gtaggcggtc aggcacagga gtcagcacat ttaaaaaaca acattcaaac ccagcacgac 36420 aagataagat caaaggtctt tttctggagt cagaattctc gtaatggaag gacccctgtt 36480 ctcactggag agagatggaa cacagcttgg ggaggaatgg ctacccaaag ggcaggaggg 36540 tggcagcaat agtgacaacg atggtggaca cttactcagt acttgctata tgccaggcac 36600 tctaagtgct tttcatgcat aatcccactg gattcccacc actgttttgt gatgtcagcc 36660 ctactttatc ccattttata gataaagaaa ttgaggctca gagaggttaa gtaactcacc 36720 aaaggtcaca cagctggcaa gtggtggaac caagatacag acccagggca ggcagtccag 36780 gtgtatcaga cagttgggct gattccatct ccctgtgcct cccagactct cctccccact 36840 gtctgctacc ttcctgtggc cttttgtggc cagctggtgt caccagcctt ctggcacaga 36900 gctcatcagc ctggagcgtc accctatgcc tggctagaat ctgtttgaca gctcattatt 36960 ctgccgagtc cttcctgctc acaggtccag agagtggaca ctggggaaag ggtggcagct 37020 aggacccagt gaacctggtg aggacctgct cagtgaaggc ttcaaccccc tggcaaaacc 37080 ctcctgtagg tggtcctggt ttctgtgtct gtgtctgtct gtcctctggt ctcctgtgtg 37140 aactgtgaca ctctgcttct tgagaacact caggagatgt cttgcatcct tgcagtttgg 37200 ccatccagag aacttccatg gcacctaggg atggagccct cactctttca ccctggcact 37260 ctgcttccag gcctgggtgg aagctgtcaa aggcagagtc cccagtgccc caggcggctc 37320 cagtactgag catggtttct cctctaagtg tcgtgcatcc atgccctcct ccacgcagag 37380 gagatcctga ggtgccaccc tgagggctct gacgccactc aagatcccct tcttgctgag 37440 aggctatagg aagtgcctct tttgggggtt tcgggagacc cttggccccc ttgtcagaca 37500 cagcactctc ttgtggatct ggctgccgga cttcaggttg gggagagggt acaatgcagg 37560 agacttgata ttcctctttg ttttcacagc tgccaaaagc ctattgaaca agaagtcgga 37620 tggcggtgtc aaggtaagtg tctccagcct ctgaacagac tggcctcttt ctccccgcag 37680 tcactatggg aattcttggc acctggttcc ccctttccca gggaatcttc ctatccttgc 37740 tagtctgctt taaaccagat gcctttgtgc tcagaacaga aggttctgct ggcctgagag 37800 ggaagtaggg aggtattttt cctggcccta gctggatggg aatgactcag gggaagtgat 37860 ccaaatcata gtttatacca gagctgaatc cggaacctga cttctacacg gatgcttcat 37920 ctccagggct tgactctggg ttttttaggt catttggtta tctttctttt tttccttttt 37980 agagcacaaa tccttttaat caaatgaaag ccaaatttgc ctgagtgatt caggcagggt 38040 atagggcttg gaacctgaaa ccactctcct tttggtcttt ttccttctct ctacaacact 38100 ttcagatccc actgagtgca acagcctcga gctttcttga cgcataggct cctcagaaaa 38160 aggcaaaggc catggtggat cacggcttgt tcccactggg tgagggagct tttcccatgg 38220 gactggggca agaggaggga cctgggaccc accaggagcc ctgctgggaa tggctgcttg 38280 gccaaggtag aggagaggtg actggggcta cccacagggc ccaagacatt ctgtagatgc 38340 tttgggggca gaaaggatcc tggggctagg gcattgggta ggagctcatg ctatcttgaa 38400 gcctcccagc ttacactcta gactagattt tcactgggcc ttttcccaag atcttgtgtc 38460 aacagctgag atacacacac aagccccgtt ccctccccgt tccctcccca ctccctcctc 38520 tttccctcat tctctgcatg cctgcttctg tgttcttccg cccctcgcag gggagcctgg 38580 gctccgcgca caccctctga catggagctg ggggcatcgt gcggtcccca agctctgccc 38640 ctgagctaca tggatggagc caggtgagga aaaggggcag gtttagttgg agagagtgtt 38700 taataagtac ctgtcagtca gatgtccacg cagcattctg ttctgagggg tacacaacag 38760 aggtgtaaga gggggtgtgg ctttcagtcg ccataggaag ggggccgcac ctggagtcag 38820 ctgagcgctg ctagtggacc cacgcgagat ggtttagtcc aggaagctca taggagagag 38880 cgtactggag aaagctgcag ggacataggt gagactcact ttgcagtttt actttctgct 38940 atatgttttc tttaaattga aaatatgggt caggcttggt ggctcactcc tgtaatccca 39000 gcactttggg aggctaaggc gggtggatca cctggggtca ggaattcaag accagcctgg 39060 ccaacctggt gaaactccgt ctctacaaaa atacaaaaat tagccagtca taatgaccgg 39120 tgcctgtaat cccagccact cgggagtctg aggcaggaga atggcttgaa cctgggaggc 39180 ggaggttgca gtgagccaag attgcgccat tgcactccag cctgggcgac agagcaagac 39240 tccgtctgaa aataaagaaa agagaaaaga aaacaacatg acatttctat aacttaaaaa 39300 caacaaatta tatttgtatg ggttctctta tacatattga tgttctctgc ccagtgagaa 39360 cacagggtgt gtggtagatt gatgtcaaaa atatggttgg atcagtctta tcaggcagaa 39420 ttggaagttt ctgtgtcaga ccatgggaaa taccataggc cattgagcag ggaagctatg 39480 gtgagagtgc tgatagaaat gatttggcaa gccgggtgcg gtggcttcac tcctgtaatc 39540 ccagcatttt gggatgctga ggcaagaaga ttgcttgagt ccaggagttt gagaccagcc 39600 tgggcaaaac cttgtctgtg aaaaaaaaaa aaaaaaatta actgggcata gtggtgtgca 39660 tctgtagtca cagctacttg ggaggctgag gtaagagaat tgcctaagcc cagggagttt 39720 gagcctgagg tgagccaaga tcaagccact gcactctcca gcctgggtga cagtgaaacc 39780 ctgcctcaaa aaaaaaaaaa agatacctgc tgtgccccta gaagttggga aggcaaaact 39840 taatctacct tttaaggtgt ttacagtggg agagacacaa ggcagctact gtttctatgg 39900 agtctgctaa ggtctcaggg aggtgtgcac ctggcaggtg ctgggggagc agacagataa 39960 acatccaaac caggacagga atcttctgga aggagatggc caggaattga gcttgaggga 40020 gtagctggat tttgctgggt taaggaggag acaggagggg agggatattc caggcagagg 40080 gaagagcgca tgtgaagata cacgaggttg aaacagcatg atgattctgg gaacttcagt 40140 atcttcttta tggctgaagg gaagagcaat tgcataaaat gagacctgaa ataaagcagt 40200 gactgttgag gtggagggga gaggatggaa aaggcaccat tacagaacag gtttctagcc 40260 aaactttcta gatactactg gtgtcaaaga tgaaggtcat gtgcagccat gtaagattag 40320 cccaaggagc cagctcaaac catgcacatc cagggcccag cttggaattc atgttctgga 40380 ggccttggct gggaggcaga atctgtgaat tttaaaaaca ctttcatgaa tccaaagcac 40440 atgaaggttt aagagtctgg taaaggcaaa attttggggt tatgtgttaa gaaagggctg 40500 gaacaagagt cggcaaagga aacagaggaa ggacagagag gtagggggaa aagagaaatg 40560 tgcagcagct gcagctcttc caggaaccct gaggatgagg gctgggcaga cacatcatta 40620 ggtaaaggct ttaaatgagg acgtgcgtgg ggaacctagc cctgcaatgt gttgtgtgtc 40680 tgaccctgat atgtgctcag taaatgagtt ttatgccaca ttcttttgag aaaagagctt 40740 caatatcatg gtgggaacca gaggccaatg atcacccaaa attaaaaggc caaccgcgta 40800 ttcgcagccg ttgtgatggg aggggttaat atttttattg aaagagtttc tgtgacaaat 40860 aatccctctt aaaacccagt agaagctggg cgtggtggct cacgcctgta atcccagcac 40920 tttgggaggc cgaggcgggt ggatcacgag gtcaggagat cgagaccatc ctggctaaca 40980 cggtgaaacc ccatctctac tgaaaataca aaaaattagc cgggtgtggt ggcaggcgcc 41040 tgtagtccca gctacttggg aggttgaggc aggagaatgg cgtgaacccg ggaggcggag 41100 cttgcagtga gctgagattg tgccactgca ctccatcctg ggtgacagag caagactccg 41160 tctcaaaaaa aaaaaaaaaa aaaaaaaaaa acccagtaga taggctaggt gtggtggctc 41220 acatctgtaa tcccagcact ttgggatgct gaggtgggct gatcacttga ggccaggagt 41280 tcgagaccag cctggccaac atggtgaaac cccctctcta ctaaaaatac aaaaagtagc 41340 cagtagtggt ggtgcacgcc tgtagtccca gctactcggg aggctgagat aggagaatca 41400 cttgaacctt gcggggggca gaggttgccg tgagctggga ttacaccact gcactccagc 41460 ctgggggaca gagcaagact ctgtctcaaa aaaaaaaaaa aggaagatag atgatcaaag 41520 aaaataaact gacaacctga aaacaaggaa gtagaactgg ataacaaatg tggaaaaatt 41580 tctagcctca ctagtatcag agaaatgcaa attgaaacaa ggtgccattt ttggactcta 41640 gttagtgatg gtagtgaaaa ccagaatggt cctttctaaa acagcctgtg tgtcaaaacc 41700 ataaaaatgc ttctacctct ttttaccctg ttaattctac ttctgagagt ttttcctaaa 41760 gaaataattc aaaataggaa aaagctaaaa gcagaaaaat gttgaacatg acattattta 41820 tagctgtgga aagattggag gctgggcaca gtggcttatg cttgtaatct cagcactttg 41880 tgaggccaag ttgggaggat tgcttgaacc caagagcttg agaccagcct gggaaacgta 41940 gtgagacccc atctcttaaa aaaaaaaaaa aaaattagct gagtgtggtg gaacgtgcct 42000 gtagtcccag ctacttggga ggctgaggtg ggaggattgc ttgagcccag gaggctgagg 42060 ttacagccag gatcacacca ctgcgctcca gcctgggtga cagagtgagg ctctgtttaa 42120 aaaaaaaaaa aaaagagaga gaagaaaaaa aagattggag acaatttgaa aagccagtaa 42180 ggagccagac acagtggtgc gtacctatag tcccagctac tcaggaggct gtcgcaggac 42240 agaattgctt gagcccagga attcgaggcc agctgggcaa catagtgaga cccccaactc 42300 ttaaaaatgt ttttaaattt aaaaataaaa agatttttta aaagccagta aatgactaaa 42360 taattatggg aaatctactt aataaactat tcaaaagtta ttaattttca tgaccgtagg 42420 gatattttaa gtgaaaaata aagtgcagaa atgttttata ttaagtgaag gaagtggtat 42480 ataaaggagt acagacaagc caggcacggt ggctcacgcc tgtaatccca gcactttggg 42540 agcccgaggc agacagatca cgaggtcagg agatcgagac cagcctggcc aacatggtga 42600 aaccccgtct ttactaaaaa tacaaaaatt agctgggcgt ggtggtgcgt gcctgtaatc 42660 ccagccactt ggaaggctga ggcaggagaa tcgtttgaac tagggagtcg gaggttgcgg 42720 tgagccaagt gcgccactgc actccagcct ggtgacagag caagattctg tctcaaaaaa 42780 taaaaaaaaa aaggagtaca tacactatca ttctaaattt ggtttgaaga aacgtgtttg 42840 tagatattta ttcagtatat aatatgtgga taaaaaaggg actggaagaa agcccactaa 42900 gtgtcaacag taacttcacc aggtgatggg aatttgagaa acttttttgc ttacacattt 42960 ttctgtattc ctatattttt catctagatt gtgcactact gttatcagaa ttttttttaa 43020 atactatttt ttttttaaag taaagcataa taccaggtgt ggcaactcat gcctggtaat 43080 cccagctact gggaggctga ggtgggagga ttgcttgagc ccaggaggtt cagcctgggc 43140 aacataagca agactccatc tcaattaaaa aaaaaagaaa agaggtaaga catgtgcttg 43200 tattattata tcttataatg atatcttttt ttttgttttt tgagacaggg tctcactctg 43260 tccccctggc tggagtgtag tggtgtgatc ttggctcact gcaacctccg cctcccgggc 43320 tcaagtgatt cttccacctc agcctcctga gtagctggga atacgggcat gtgccaccac 43380 gcccggctga tttttgtatt tttagtagag acggggttgc ccaggctagt cttgaactcc 43440 tgagctcagg tgatctgccc gcctcaacct cctgaagtgc gggggttaca ggcatgagcc 43500 accacgcctg gcctataatg atatcttaaa agattgcttt cttttttttt tttttttttt 43560 ttttttagac ggagtctcac tctcacccag gctggagtgc aatggcatgg tcttggctca 43620 ctgcaacctc cgcctcccgg gttcaaacaa ttctccaacc tcagcctccc aagtagctgg 43680 gactacaggc gcgtgccacc acacccagct aatttttata tttttagtag agacggggtt 43740 ttgctatgtt ggccaggctg gtctcgatct cctgaccttg tgatccaccc gcctcagcct 43800 cccaaagtgc tgggattaca ggcatgaacc accgtgcccg gccaattgca ttttttaaaa 43860 agactggaag attgctagga gtattagtgg ttttcccatg ccccttctct gttttccaaa 43920 ttgcttgtat tgtggctgca gtccttttat aatatgaaac aggtaaataa caacttatgt 43980 tgtggctgca tcaaaggggt gagaaacgaa aaggagagga caaagcaaga tgtgcagagt 44040 tcgacctttc caggctctct caaagtcaag gttttgatca atgttatgag ggaggcctgt 44100 gaagtagctc agatggtctt gagctttcag catcatggat tcttctttta gatcccatct 44160 tcccttccca actccccctt cctcaattcc tactgcttaa gtgtccatag ggcgatttct 44220 ttttcactgt tcagaagctt tctgcaagat gttcaaaata ctagcattgg tttgagcagc 44280 tagtctgtct tgtgttcttg atttggggga cttagcttct atttagattt ctttgaagct 44340 ggatgccagt gacccagggt ctatggaaga gtaagagcca cttgtgagga tgactgaaga 44400 ggccacaaac tctcagatcc tgagagtgta ggacaacttg tgccttctgc tagtcccagg 44460 ccagaatggc catcctatct ttaaaaaaga aagcaagcaa gaaaaacgaa aggttatagt 44520 tatttcccta agtactattt gaattatttt gttaaattaa gtatgagaaa gaggtttgaa 44580 cgcttttcca gcttaaaatt taaaataaat atacagtttt taagtaaaag tgagatatga 44640 ttctttagaa atcatctggc atttagccag gcatggtggt gtgcacctgt agtcctagct 44700 actcaggtgg ctgaggcagg aagatccctt gagcccagga ggttgaggct gcagtgagcc 44760 atgatcatgc cagtacttca gcccgggcaa tagagcaaga ccttatctct aaaaaaataa 44820 taaaaagacc tcacatttag acaatgtggt agtgtgctgg ttcagaagga gcccagctat 44880 gcatggctaa gggcaaatcc ctgaatggag aaggaaattg aaaaatgttg actaacctga 44940 gaaacagtct ttggaaaagg gtgatctcag gttctcatgc aggacaattt aggaaaaaga 45000 gagcaagcca ggagaaggct gagaacttat tccccattag tcaaaaatct gctttaagtc 45060 aagatcctgc aatggccttt cacaacaagc ccctgaaaat cagcagaaca aagactgggc 45120 ctggtgagtg agtgcctacg cagagttctt gctgccgtga ttcagtgcaa gttagaaacc 45180 tgtgctcttc tttagcctgg ggaaaaacca aagtcagcaa acccagctca actcagcaaa 45240 ctttcgtcgc ctgtatgcta actataaggc atgttgctag gtactgtgga aattgtaaag 45300 acacataaga taggaacctt cctgaaagca gtaacacttt agttgggtaa agggataagg 45360 agatatacac acacacacac acacacacac acacacacac cccactactt atatatatga 45420 atataaggga actccttctt tttgagggat gattttcgaa gtaaaatatc atatttgagc 45480 atatttaaaa ggccactgta aggctgtgtg cggtggctca cgcttgtaat cccagcactt 45540 tgggaggccg aggtaggtgg atcacctgag gtcaggaatt cgagaccagc ctggccaaca 45600 tggcgaaacc agtctctcta ctaaaaatac aaaaaaaaat cagtggggcg tggtggcggg 45660 cgcctgtaat cccagccact caggaggcta aggcaggaga attgcttgaa ccagggaggc 45720 ggaggttgca gtgagccgag atggtgccac tgcactccag cctgggcaac agagtgagac 45780 tccctaaaaa taaataaata aataaataaa taaataaata aataaataaa taaaaggcca 45840 atgtaaaaga ggcctaacta tatttaggtt tttcttttcc tttaaatcta attctaaatt 45900 atggaccatt gtcaatattt gtagcctctt tcgttgatta taataataat ccctgaaaat 45960 gccttctaaa gaatgctggc cgcttgaggg caggagcagt ttatcagctg tgtttacctg 46020 aaacagccct cagtgtttgc tgggcattgt taaatgaatg tgcaaaagtt gaacgacaga 46080 cggacatatt acagggggac cttaccccca gtgagctaat gatgacattg ataattaccc 46140 ttcatttttt agacacagtc ttctgggata tattttcagt gttccacgtg gtcttcatct 46200 tgatgcgtct gtttcacatg tgaacgtaaa gttcgtgagc atctagttga ggctgaggaa 46260 tcactgcttt caacattccc tgtggcttac atccctgcat ttttatgatc actgtagttt 46320 taatcactgg cactcctgtg tttctatttt ccacgaattg caaaatgcaa taaaaaattc 46380 aaatattgta aacaagcatg gctatactga caaaggaagg ccaacattta actgctaggt 46440 gattttcaaa agctcagcat ctttatgtaa aaagcatagt agggatgcag cgaagtcaga 46500 agtcaaattt tattagagct gaggagagcc tgtagtagct tttgcttttt ccctggtggc 46560 tgctcacttg aatttcagac agttctagta atgagagaaa ataaataaca ttacagggtg 46620 agctaaccct atgaacccag acctgtaaat ttgtagcaaa atgatactta acctcacaga 46680 cttgtgtctt aatctcctta agaggctttt tttgagcaag gctgagacat ctcagaagat 46740 actaaatctg tgtctatgaa cctgaccaca aaagagttct tccctcccag ggtctggagg 46800 gtgtgagtgc ctgtcggtcc gtgtgctgtt taaccctctg gtgctggact ccgggtctcc 46860 ctccgctctt ttctccctga tgcagagccc acactggtgc gctaacctgc agcgtctctg 46920 tgcttctctt cttacctcct ctttcccctt ctctttccct cttgctgtgg tgtgtccaga 46980 aaaggaagtc gagttccagc gtgcacctaa tggtgagcct tgcctgccca cccatcgccc 47040 actccatgct gcctgtgccc gcctgccagc cacgcaaacc tgttctgcca cgtgcgtgtg 47100 cctcactcat cctcactgca tgtctgtgct gtgtgggcag gtgtggcctg tcctgccagg 47160 cgggggccat tgcccaaggt cacccagtag cctaaaaagt ggacattgga aggggtggta 47220 cggcaccccc tgctgtggag cttggacaga ccccagcgac ccagggtagg atgtgaagct 47280 ggtagggact tggggcaagc aagggagaga ccctcactct cttgtcaccc agaaggagag 47340 gccctgcttc ccaggcatga ggagctgctt cctacagact ggcagctgga gggcaactgt 47400 gtggtgggca gaggagctgg ttgcaggctc ccacttgtga gtctcgctct cctggctctg 47460 cccccgtgca aatcccattc tctctagctg tgcccagtgg tttattctgc ccacccagcc 47520 ctcgggggac agctaactca tctttctcac gggacactgg gcaccaaggg caacacagca 47580 gcctgagtca ttatgaaacc atccattaaa accagaggtg ggggccgggc gcgatggctc 47640 acgcctgtaa tcctagcact ttgggaggcc gaggcgggtg gatcacaagg tcaggagatc 47700 aagaccataa cacggtgaaa ccctgtctct actaaaaatg caaaaaatta gccaggtgtg 47760 gtggtgggcg cctgtagtcc cagctactca ggaggctgag gcaggagaat ggcgtgaacc 47820 caggaggcgg agcttgcagt gagccgagat cgcgccactg cgctccagcc tgggcgacag 47880 agctagactc cgtctcaaaa aataaataaa ccagaggtgg ggccacttgg gtgacatccc 47940 agccctctgc aggttttgtg ggcaccctgg agtccttgcc ccctgtgagg gtcttggcct 48000 cagctgggat ttacaggtag ggcagccctc tctaaccaac cccgaacagg tcagcatcat 48060 tcactgagct aggtgggctt tgcttcttgg tgggaatgag agacagcaga gctcccgtga 48120 gtttagaccc accgtctcac tactcctggg ccccctcttc tctagcctgt cgcagtctgt 48180 ggagtcttgt tcagtggagt cacttggtgc ctggcttgag gttccatgcc tagccctggg 48240 tttggggatg tctgagccat tgacagcaag ctggcggtgg acggcttcag gtctggtcca 48300 agaggcctcc aggcaagaag taggacagtc aggatgcttt ctgtgtatgt cctaggagag 48360 aagacacaca ttctagctgt cgatgtatca tctgtgccct gtgcagggat ggtagccaca 48420 catttgtctc actgcctatt gaagaacttg caggcatcag gctgctcctc agtggccccc 48480 aaccccactg gaactcagtg agatggagta cgctggttag ggaactatca gaggcaaaga 48540 acatcacatg gatatggctc cctgccctgg agatcagcct tcttcctttc ttccatcttc 48600 cccttgcccc tcccttgctg tgcccctccg tgtaatgttt ttgtttgttc gtttgctttt 48660 ggttttttga gatggagtct tgctctgttg cccaggctgg agtgcagtgg tgcaatcttg 48720 gctcactgca atctctgcct cccaggttca agcaattctc ttgtctcagc ctcccgagta 48780 gctgggatta caggcatgtg ccaccatgcc cggctaattt ttgtattttt agtagagacg 48840 gggtttcacc atgttggcca ggctggtctt gaactcctga cctcaggtga tccacccgcc 48900 ttggcctccc aaagtgctga gattacaggt gtgagccacc gtgcccaccc acccaccatg 48960 tagttttgaa aggcaaggag atatccctgg tggtcatggt gctgttggga atgttggcct 49020 gtgtgtggcc tactctgtcc tgggggctgg attctgggac tacagctaca gccccgctgg 49080 gtttcacctg cccctccccg gaacactgcc cttctagctg atcaggccta agatttgtca 49140 gacaaaaagg tgaacagcac agtcctgact ctgctccctg aggtcagtga atgcattttg 49200 tgtctgaaag ggacttccac ccccatcctc tggacaccat ctcttaggcc aggcatactt 49260 ttcttttctc cttcctcttt gtttcaggct tcgagctggt gttgtaagaa ggaaatacag 49320 gtgctgggtt gaaagtgcag caggagactg cccacagata ggggaccaga gtttctgaat 49380 tttgttctgc tttcttataa actacccccc tttttcctgt acagtgggaa gaagatcttg 49440 aacttctttg ggtcaggtgt ggattttgca atgacctggc acctggcata agcagagatt 49500 tctggaggga tgctttaaaa caaggctttg ggctggtccc accttgaggg tgcccccaga 49560 gctaggtctc tgggccccac aaatacttcc tctgatcatc tctctagcca tcgctcccat 49620 ctacacagcg ttatggaggc cacctcaggc ctacctcctc caggccagac cagggggcaa 49680 gggaggtctg ggagttgaac ctgagtggcc ttggggactc tggaggaact aaaccatctg 49740 ttttcttgtc tcagccacag agcaacaaca aaaacagtct cgtaagccca gcccaagagc 49800 ccgcgccctt gcagacggcc atggtacctc ctgactacag cttctccgcc tctgaccctg 49860 gctgcctcct gccccttccc tcttcctcct cttgtgcccc ctccctggcc tcctggcctg 49920 ttcctttctt ggtccccata gaactgactg ctttgtgtgc cgccctgtat gccccttccc 49980 cttcattgtc ccgcctggcc gcgctccatc ccgcatggca gaagtgctgc tcctgctcct 50040 gctcctttcg ctggtggggg gaagagtgat cagggctctc agctgaacct cccaggccca 50100 gcccaggacc cctagtgggt ctgctgtggg ggctgggaag gtgagttgct taggaaagga 50160 gagggtagga gctttcttgg gacctgaaca tcagttcttg gaggccccct tgtaaaacct 50220 gcctcagcct ctcctttgca aagccagaaa caggaaagag ggctggggtc cccacctctg 50280 gatggtgctg aggtctccag gctcctggag tgcctcatgc tggctaagtt ctctctgggc 50340 tcctccaggg gttctgtgtg ctcttggagg tccctctgct agtggtggct aactagagag 50400 tcagcagggg ggtgactggg aaagagggag aggtgatgtt gcctgctact cccctccttg 50460 cggaccctca taccacgtga cgtggcggcg tggggccagg aactagggaa ggcagaaggc 50520 gggcgcagtg ggcagctctc tgggctcagc ttgctgaggg ggcctcctgt cctggctctt 50580 tctgggagac ctcattcttc tgcccatgtt cctgcctcac acattccccg tgatgaacgc 50640 tgtgggcggg gcccggcctg tgccctcagt cccacagctc ctctagtgta cctgccccgt 50700 gggaacccca tgtggaaaga gccctcagaa ctgacaggaa tcagggacag aggcccttgc 50760 tgtcagcctc ctgggcacct gcacctgcca ggcctctctt tcttaccagc ccagtgctgc 50820 tgccaaaatc cagggctatc ccagctgccc gggaccccag ttgagccggg atattttgtc 50880 ttctggagat ggctggtggg caggcctcag tggtcatcat agggtctgcg ggggtcctgg 50940 ggtgcaggtg gggctcctca gggaagagcc atagtctgtc cccaagtcgg aagggtaatc 51000 ttcatcttct ctcacaggag ccacaaacca ctgtggtaca caacgctaca gatgggatca 51060 aggtgagtgg ctcctgagcc tgcctcctgc tttccaggtc agcaggagac aggtgggctg 51120 ggtcccaggg gtctacaggc tgcaccctga ggccaaggtg tttgcagagg ctcagctgaa 51180 ggtagcctgt gcccacagtt gctccatgct gaggaagggc attatacctt acagagctca 51240 ggctttgcag tcagacagac ctggtctgaa tcctggccct gcaccttagt atcctttatc 51300 tgcaaattgg ggatgataat aatagaatct tcctccatat gtcggaagtt taaatgagag 51360 taaacgttca ctgaaaaaat aggcaagagt atctccagac cctggagcgt tctccatggc 51420 ctgacccctt tgtgcccttg atgttttcac cagcattcct gaacatctgt taagcccaga 51480 taccatccat ggctctggct tacagaggtg acaagacaaa ttatctgttc aaacggtggg 51540 tgggatggga ggcagataaa aaaccaataa gcaaacagat aagataagct gggcaccgtg 51600 gctcacacct gtaatcctca cactttggga ggccaaggtg cgcagatcgc ctgagctcag 51660 gagttagaga ccaccttggg caacatggtg aaaccctgtc tctactaaaa tacaaaaaag 51720 taggcaggtg tggtggcgcg tgcctgtagt cccagctact tgggaggctg aggcacgata 51780 attgcttgag cctgggaggt ggaggttgca gtgagctgag atcacgccac tgcactccag 51840 cttgggctac gcagtgagac ttaatctctc aaaaaaaata aataagataa aatctaatgt 51900 caataggtaa tctgaagaaa atggcagaaa gtagagagag ggccaggtgc ggtggctcat 51960 gcctgtaatc ctagcacttt gggaggccaa ggcgggcgga tcacttgagg tcaggagttc 52020 aaaaccagcc tggccaacat ggcaaaaccc catctctact aaagatacag aaattacctg 52080 gggatggtgg cacatgcctg taatcccagc tacctgggag gctgaggcag gagaatcgct 52140 tgaacctggg aggcggaggt tgcagtgagc tgaaatcgtg ccactgcact tcagcctggg 52200 cgacagagca agactccatc taaaaaatga aaaacagaaa aacctcacca aactagacag 52260 agagaacagg gccttgaatt aagtagtcag gagagggctt ctttcaggag gtgatatctg 52320 agctagaaac tgaatggtgg gtgggaagga ggcagccagg ccagctctga ggctgagtgc 52380 cctaagcaga aggaactgaa gctcagatgt ggcctttgta atcaagcaga gggaagagca 52440 aagtgagacg gggagaacca taggagagtg atgaggttgg agaagcagca gggcctgcta 52500 cagaggccct tgtaggagtt tgcattttct tccagcagca aggagaagct attgggagtt 52560 cttagcagga gtaacagaat ctagttgaca ctttaaaaca ccactctggc ctcatgatca 52620 agaactctag ggaggcccgg gcgtggtggc tcacgcccgt aatccctgca ctttggaagg 52680 ccgaggcgag tggatcagca aaggtcagga gctcgagacc agcctggcca acatgatgaa 52740 accccatctc taataaaaat acaaaaatta gccaggcatg gtggcaggca cctgtaatcc 52800 cagctactca ggaggctgag acaggagaat cacttgaacc cgggaggcag aggttgcagt 52860 gagccgagat catgccattg cactccagcc tgtgcaacaa gagcaaaact ctgtttcaaa 52920 aaagaaaaac tctagggagg aggtaagtgt ggaagttagg gagaccatga agctgttatc 52980 atggttcagg tgtgagatgc tggtggcctg gagtcaggtt gtagctgtgc attggaagtg 53040 aagaggtaag acatggggtt tactttggag gcagaaccag aagattttat tttagattgg 53100 gcgatctgaa tataagggaa aaagagaaag agaaggattg aggatgactc caggttttag 53160 cctgagtaac tgggtagatg gtggcattta ccaactgggg gaagactagg gaggggattt 53220 gggaagagtc agacagccag ggtggaagca gaaccttcca caattcctcc ttgcacctct 53280 tgtaggagca gaaactctgc ttttgttctg ctttgctcct ctggcttcca agggatggag 53340 catatagaaa catgttcttt ttggcctaca gggctccaca gagagctgca acaccaccac 53400 agaagatgag gacctcaaag gtaggtgctg gcccttggag ggggaaggac tccagcagtg 53460 acccaggtac ctgggctcca atggggcacc tgccttttct gtccccagaa ctgggaatgc 53520 tggctcctat gcccctagga gagggcttgg tataaaagct actttccacg agccaagata 53580 tgaggcccct gtctggtgtt gctgagttgg gcaagaggct tctcttcttt gaccccaagt 53640 ctaaaatagc taagctagag attctccagg ggccagggct cagagaactg ttcctgttgc 53700 tgataatgat gtgccatcca agaacagggg taccccaagt ccctgccgaa gtagcctgta 53760 agtgctatga gtcataaata gagtgaccaa tcactcctgg ttttcctcgg acacagaact 53820 tttggtttta agactgtgat gggccaggag tgctggctca cacctgtaat acccagaact 53880 ttgggagggc cagggcagaa ggattgcttg agaccaggag tttgagacaa gcttgggcaa 53940 catagcaaga ccttgtctct attaaaaaaa aaaaattagg aacaaataaa taggccaggt 54000 gcggtgactc acacctgtaa tccccacact ttgggaggcc gaggcaagtg gatcacttga 54060 ggtcaggagt tcaaaaccag cctggccaac atgatgaaac cccgtctcta ctaaaaatac 54120 aaaaaaaggc cgggcgtagt ggctcacgcc tgtaatccca acactttggg aggccaaggt 54180 gggtggatca cctgaaggtc agaagttcaa gaccagcctg gccaacatgg tgaaactcca 54240 tctctactaa aaatataaaa aattagccag gtgtggggca ggtgcctgta atcgtagcta 54300 ctcgggaggc ggaggtggga gaatcgcttg aacctgggag gtggaggttg cagtgagccg 54360 agatcacccc attgcactcc agcctgggca acaagagcga aacttcttct caaaaaaaaa 54420 aaaaaaaaaa aaaaaattag ccgggtgtgg tggcggggtc ctgtaatccc agctactcgg 54480 gagactgagg catgaaaatg gcttgaaccc gggaggtgga ggttgcagtg agctgagatt 54540 gcaccactgc actccagcct gggtgacaga gcgagactct gtctcaagaa aaaaaaaaaa 54600 aaaaatatat atatatatat atatatatat atatatataa atataaaacc cagatagtcc 54660 tgggaacact gggatgagtt ggtcactcta gtcttaagat tttggcctga atgatggagt 54720 tggaactaat ctgacaaccg tgaggccaca tttggtcatg tcctggtggg cccgtaagga 54780 ccactagcct aagcttgggc ctggctagag tgccagggcg gtgggagggc atggcaggct 54840 ggacccccgg gaatctctgt cctgctcttt gattgggcct cctggaattg ctccctttgc 54900 ctgaattcag taagtgacct tgggccagga catcagaaaa gacagaggaa cactctagga 54960 cagagctggg agagcatgcc ctgggtggca agggggcacc aaaccttttg gaaccaaaaa 55020 aaatagcaga aagctgcgag gaagtgaatc atagtagctc caggcccctg tgagtgaggt 55080 cagatcagtt ttgattccgg cactgctggc aacataggag gcgctgtcac tgctgggctc 55140 tggaccctgt ggcctggccc cctggaacat cttccccggg atcaggggtc cttggacagg 55200 ctgttgtaag gctcgtctgg aagccacagc ccaggtctgg gcacctgcct ggtgccctca 55260 gctgggaggc ctctctggca gaggcggcgg cgtgggatgt cgtccagtgt ccacagcagc 55320 ctgaggcgag gcgtcccctt gccccggctc tacagcgcca tgggctcggg gcctgtctgg 55380 cttgctcgct cacctgcctt gttctgtttg ttttggctgc tctgccttgc cctgccctgc 55440 cctgccctgg ctggctagct gccccgctcc gcactgggaa tggcagctcg gtgcctgaag 55500 gacggagctc ccgggacaga acagccccct ctgcaggcat gcagccccag ccttctctct 55560 gctcctcagc cagtaagtgt gagggaggca cattctggct tccgtctccc tggctcgtcc 55620 tgaagcccct cagggacccc caccacagct gtcagtccca cccacctgcc cgtggtagta 55680 agctctggga gcatggcctc tgctgggggt ggggggtaga ctggaggtgc tgttgagacc 55740 aggcaggggc ccctgagtct ggggcccaaa gaaatatgag aagtgtgggt ggaaaaacat 55800 ggcctgggat gaggggagta gaaagccccc aggatgtgca gtgggccttg cctcagcgct 55860 gagccaggaa gaagggcaga gtcggaagtc aggtctgtgg gggtgggagt gggatgatgg 55920 ggaaatcgtg acagcgagga actgtgttgg ggatgtagtg cttcctgagt ctcagcataa 55980 cagtattaag agcatggggt cagaggcaag atagatctga gtttaaatcc cagctacact 56040 gccttcaaga gtgtgaagtt taacctccca gagctgcagg ttccttatct gtaatgtgga 56100 aataaaatgg cacgcacctc agagccttgt tagataaaag acaaggcagt aggaagtctt 56160 gatacggtgc ctcgatgggt tatcagtagc tcatcctcat atttctagtt acgtctgtgc 56220 tggaggatgc ctttgtctgc tgcttttcct cccaccatct atccttgcag agtttctaag 56280 cacaaccctc ttcgcccgtg gggccccagt caggtcatcc agatgggtct ggtggggttg 56340 gagagggtgt gtgtgttgtg ggtgcacacc tgcctgctgc ttttggaagc cgatcgaact 56400 ccttgcttcc cttaacctgc tgcttgctca cctggagctg tggcctagcg gggctgacgg 56460 ctgtggggcc ccctcctgga tgtgcctttg gctgcgctgc cctgtcccaa ctgtgctgct 56520 tggctgtgct ggcccggctg ggccgtggtg gtgctgttct aacgcttgca gttgtcttgc 56580 agccttttgc tcctgtgagg aaagggttgt ggcctggccc cgcccagggc tcgggttagg 56640 atgagcccaa gctcaaccca agctctccct taccctggtg gcagcccctg ctggtagtgg 56700 cattccctat aagagaagcc catgccggca ggacatcacc agctgtccct tggctttgga 56760 tgggttgggg aggaggcctc tggagggcac cacctctgcc tgcctgtcag tctgagccct 56820 gtctggtttt cctgaggaac acgtcctggc aatgagagct ggtgtgaaat gtgcagcttt 56880 cccaagcctc gagaggtaaa tggagcagcc tctctggtac aggctgtccc aagtttttac 56940 agttctggga tcatttctcc cagaaaagcc ctgtggagtt gagcagtggg aagcatccat 57000 cctagggttc tgatggtctt ttggcacccc agccctagct ggattctgct gtcaggctac 57060 ctgtcaccca gggctgggtc ctggccactg aatgagggct acgagtgggg gtggtgattg 57120 agacctgact gagccccttc aggtgagaga agtaaattgg gggtggaagc ggccttattg 57180 ggagatgctt gtgagagagg ctgctcatac aggggagggg ctcacagcat tcacgatgta 57240 ccaggctcct cacctgttaa aggcaagcgt gttttctgca acctggttgt tgatggaaag 57300 ggaggcaaag gccaaagaac cataactaat ggctgggctt caggagaaag tggtcattgt 57360 ctctgcagac tgcagagagg gagacgggag ggaaggtgtg ttcgctcttc ctgccaaggg 57420 ccctagagac agagaagagg gatgtctttg tcataagcga tcacagggga ctcctgagga 57480 ctggggaggg ctctctgtaa cttgggaggt tccccagtag gtaaattgat ggatttttct 57540 cccccacagt gcgaaaacag gagatcatta agattacaga acagctgatt gaagccatca 57600 acaatgggga ctttgaggcc tacacgtaag tagagaccca tttttttttg tgacctaagt 57660 catctcccaa ggccttccct gcttccagac aacaattagg accctgggga aagggaggtt 57720 ggaccttggg caaagtatct gagttaagcc ctctcctaaa ctgggagccc ttccaggtag 57780 attccctgag ctcacccatg gtatcctggc agtgggccga aagcacaggg ctgagtggct 57840 cagcaggcag gcctggaaga tctttgctgt cttgtctggc atggccacag gtagcctgct 57900 gctactggat agacaccgct gataaggaag gaagacaagt cactccatag aagcctgata 57960 ggctgctttt ttttttctcc ctgtaggaag atttgtgatc caggcctcac ttcctttgag 58020 cctgaggccc ttggtaacct cgtggagggg atggatttcc ataagtttta ctttgagaat 58080 cgtgagtggg ttcgtgctgc tgatatactc ctgcctgccc ctttacccct ttgtctctgt 58140 ctcctgctca ccttctcatc ccagttgccc acttttccct tatttgacct tcgtgctgca 58200 ctcctactct gtatgcttgt ccccttgtgc cccgatggtt gtagacaggc acctttgaag 58260 gccctgctcc tgagctccaa gtgccattca ttctgcagct gctttgtggc agtgccagtc 58320 accacaatca agctcactta tttcttgccg ggcgcggtgg cttacgcctg taatcccaac 58380 actttgggag gctgaggctg gcggatcacg aggtcaggag atcgaggcca tcctggctaa 58440 cacggtgaaa ccccatctct actaaaaata caaaaaatta gccgggcttg gtggcagtgc 58500 ctgtagtccc agctactcgg gtggctgagg caggagaatg atgtgaacct gggaggcaga 58560 gcttgcagtg agccaagatc aggccactgc actccagcct gggcaacaga gcaagactcc 58620 atctcaaaaa aaaagaaaaa attatttaag cctcacctct ttccaagacg gattggaagg 58680 aaaccctttg agattaggtt gagatgatct cagcacataa gaactaagct ctgtgtctgc 58740 aggtttcaca atagaggaaa ttaaaaccag gataagaatg tgcaaaccag ggcactgttg 58800 gtgatttgcg agatcggaag ttgtggctag aatcttcctg actatggagg aaggcagacg 58860 tcttgtatag ggggtggggt gtacattctg gacagttcgt ggaaaataag gggataagaa 58920 gctgaatcat caccccctcc catctttctc tctgctctat gagaccctcc ccttccttat 58980 ttttatctct tcccacttta tgctgggcct tccctatcct gccctgagtt atagttagtc 59040 actaacttct ccgctggctc ccacccttat cacatctcag ctacatatat aaactctctg 59100 ttatctaagt aattctatta gccagaagca attccagagt ttatattagt actaggaagg 59160 tgtcatgtag cccctgtcta acatttgaat tgaactaaaa tgtgaatctc aataaaagca 59220 acacagtttt cacagcatat gctgataatg gcaatccaac ttcttttgcc ttttccccag 59280 agaatcctgg gaatatcctg agcttggtgc tttgatgatt ctatttcagc tttggtgcct 59340 taaaaaaaat tacaaatcaa ttttgaatgg tttaagttca tgattttgtt ctgcagccct 59400 agctaggggt gagccaagcc ttatgaaatc taaactcagc ctaacagaat agaaaatcta 59460 taggctttag ttaagagtca catggtcctg agttcaggtg tgtgatttga gcaaattatt 59520 ccttgagcct atttcctcat cttataatga agaaaatatt atccaccaag aaatacagct 59580 cgggcatgta aaaccccagc acaatgcctg attaaaagcg cagcaggtac tgtcactgtt 59640 acccatcttt ctgttccttt tggataaagg agactaatgt aatgtggcat cctggcctct 59700 ggagggcgtt caggggttcg ggggtggggg ggggcggtac ttggagattc tgggagtggt 59760 tgcttgggag atggtaagac ttggaagtgc aggctgggag gaaaatgcag gtgcccaggc 59820 ctgatgtcct cttacctacc ccaccctgcc ctgcagtcct gtccaagaac agcaagccta 59880 tccataccac catcctaaac ccacacgtcc acgtgattgg ggaggacgca gcgtgcatcg 59940 cctacatccg cctcacccag tacatcgacg ggcagggtcg gcctcgcacc agccagtcag 60000 aagagacccg ggtctggcac cgtcgggatg gcaagtggct caatgtccac tatcactgct 60060 caggggcccc tgccgcaccg ctgcagtgag ctcagccaca ggtgcacctg gttgacgggg 60120 gagaggggct ggaagggcct gggataggtg gggtcagagg aagaagagaa ggctgggagg 60180 tggtcctggg agaggaggtg tgggccgtcc cagaggactg gcaaagcctg gcagaatggt 60240 tgcaataagt tatgcttgga aatcagacag actagggtct ggctccgtga ctccaaattg 60300 gatgacctca gacaggttac ttcccctccc taaactgttt ccttagctgt caaagaaagg 60360 cagagagtgg tgcctacctc atttaatcat tgtgaggatt aagtaagata ctataagtaa 60420 agcacttagt tagtgcttag caaatgggag gcagttttgt atttaagcat tagcttcacc 60480 cactttcccc accttctcag gccgacttgg ccatgtgttt agcgtgctaa agtcgctgga 60540 actcatctgt gtgctcattg tcctctgttc tgttaccaca ttctgtcctg tttgacaggg 60600 gctttaggag attccagccg gaggtccaac cttcgcagcc agtggctctg gagggcctga 60660 gtgacagcgg cagtcctgtt tgtttgaggt ttaaaacaat tcaattacaa aagcggcagc 60720 agccaatgca cgcccctgca tgcagccctc ccgcccgccc ttcgtgtctg tctctgctgt 60780 accgaggtgt tttttacatt taagaaaaaa aaaaaagaaa aaaagattgt ttaaaaaaaa 60840 aaggaatcca taccatgatg cgttttaaaa ccaccgacag cccttgggtt ggcaagaagg 60900 caggagtatg tatgaggtcc atcctggcat gagcagtggc tcacccaccg gccttgaaga 60960 ggtgagcttg gcctctctgg tccccatgga cttaggggga ccaggcaaga actctgacag 61020 agctttgggg gccgtgatgt gattgcagct cctgaggtgg cctgcttacc ccaggtctag 61080 gaatgaactt ctttggaact tgcataggcg cctagaatgg ggctgatgag aacatcgtga 61140 ccatcagacc tacttgggag agaacgcaga gctcccagcc tgctgtggag gcagctgaga 61200 agtggtggcc tcaggactga gagcccggac gttgctgtac tgtcttgttt agtgtagaag 61260 ggaagagaat tggtgctgca gaagtgtacc cgccatgaag ccgatgagaa acctcgtgtt 61320 agtctgacat gcactcactc atccatttct ataggatgca caatgcatgt gggccctaat 61380 attgaggcct tatccctgca gctaggaggg ggaggggttg ttgctgcttt gcttcgtgtt 61440 ttcttctaac ctggcaagga gagagccagg ccctggtcag ggctcccgtg ccgcctttgg 61500 cggttctgtt tctgtgctga tctggaccat ctttgtcttg ccttttcacg gtagtggtcc 61560 ccatgctgac cctcatctgg gcctgggccc tctgccaagt gcccctgtgg gatgggagga 61620 gtgaggcagt gggagaagag gtggtggtcg tttctatgca ttcaggctgc ctttggggct 61680 gcctcccttc ttattcttcc ttgctgcacg tccatctctt ttcctgtctt tgagattgac 61740 ctgactgctc tggcaagaag aagaggtgtc cttacagagg cctctttact gaccaactga 61800 agtatagact tactgctgga caatctgcat gggcatcacc cctccccgca tgtaacccaa 61860 aagaggtgtc cagagccaag gcttctacct tcattgtccc tctctgtgct caaggagttc 61920 cattccagga ggaagagatc tataccctaa gcagatagca aagaagataa tggaggagca 61980 attggtcatg gccttggttt ccctcaaaac aacgctgcag atttatctgc acaaacatct 62040 ccacttttgg gggaaaggtg ggtagattcc agttccctgg actaccttca ggaggcacga 62100 gagctgggag aagaggcaaa gctacaggtt tacttgggag ccagctgaga agagagcaga 62160 ctcacaggtg ctggtgcttg gatttagcca ggctcctccg agcacctcat gcatgtccca 62220 gcccctgggc cctagccctt tcctgccctg cagtctgcag tgccagcacg caaatccctt 62280 caccacaggg tttcgttttg ctggcttgaa gacaaatggt cttagaattc attgagaccc 62340 atagcttcat atggctgctc cagccccact tcttagcatt cttactcctc ttctggggct 62400 aatgtcagca tctatagaca atagactatt aaaaaatcac cttttaaaca agaaacggaa 62460 ggcatttgat gcagaatttt tgcatgacaa catagaaata atttaaaaat agtgtttgtt 62520 ctgaatgttg gtagaccctt catagctttg ttacaatgaa accttgaact gaaaatattt 62580 aataaaataa cctttaaaca gtccattgtg ttactgctgt tggaggttta cggccagagg 62640 cgtagatttt agcagcctgg gttaccaggt tggagagagt acctcctcct actccctttg 62700 ggtacttttg agaataaaac ttcctcatgc ctgtaatccc agtactttgg gaggccgagg 62760 cgggcgaatc acgaggtcag gagttcgaga ccagcctggc taat 62804 4 556 PRT Human 4 Met Ala Thr Thr Ala Thr Cys Thr Arg Phe Thr Asp Asp Tyr Gln Leu 1 5 10 15 Phe Glu Glu Leu Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Val 20 25 30 Lys Lys Thr Ser Thr Gln Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys 35 40 45 Lys Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile 50 55 60 Cys Arg Leu Leu Lys His Pro Asn Ile Val Arg Leu His Asp Ser Ile 65 70 75 80 Ser Glu Glu Gly Phe His Tyr Leu Val Phe Asp Leu Val Thr Gly Gly 85 90 95 Glu Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp 100 105 110 Ala Ser His Cys Ile His Gln Ile Leu Glu Ser Val Asn His Ile His 115 120 125 Gln His Asp Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu 130 135 140 Ala Ser Lys Cys Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu 145 150 155 160 Ala Ile Glu Val Gln Gly Glu Gln Gln Ala Trp Phe Gly Phe Ala Gly 165 170 175 Thr Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Asp Pro Tyr Gly 180 185 190 Lys Pro Val Asp Ile Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu 195 200 205 Val Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Lys Leu Tyr Gln 210 215 220 Gln Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr 225 230 235 240 Val Thr Pro Glu Ala Lys Asn Leu Ile Asn Gln Met Leu Thr Ile Asn 245 250 255 Pro Ala Lys Arg Ile Thr Ala Asp Gln Ala Leu Lys His Pro Trp Val 260 265 270 Cys Gln Arg Ser Thr Val Ala Ser Met Met His Arg Gln Glu Thr Val 275 280 285 Glu Cys Leu Arg Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile 290 295 300 Leu Thr Thr Met Leu Val Ser Arg Asn Phe Ser Ala Ala Lys Ser Leu 305 310 315 320 Leu Asn Lys Lys Ser Asp Gly Gly Val Lys Pro Gln Ser Asn Asn Lys 325 330 335 Asn Ser Leu Val Ser Pro Ala Gln Glu Pro Ala Pro Leu Gln Thr Ala 340 345 350 Met Glu Pro Gln Thr Thr Val Val His Asn Ala Thr Asp Gly Ile Lys 355 360 365 Gly Ser Thr Glu Ser Cys Asn Thr Thr Thr Glu Asp Glu Asp Leu Lys 370 375 380 Ala Ala Pro Leu Arg Thr Gly Asn Gly Ser Ser Val Pro Glu Gly Arg 385 390 395 400 Ser Ser Arg Asp Arg Thr Ala Pro Ser Ala Gly Met Gln Pro Gln Pro 405 410 415 Ser Leu Cys Ser Ser Ala Met Arg Lys Gln Glu Ile Ile Lys Ile Thr 420 425 430 Glu Gln Leu Ile Glu Ala Ile Asn Asn Gly Asp Phe Glu Ala Tyr Thr 435 440 445 Lys Ile Cys Asp Pro Gly Leu Thr Ser Phe Glu Pro Glu Ala Leu Gly 450 455 460 Asn Leu Val Glu Gly Met Asp Phe His Lys Phe Tyr Phe Glu Asn Leu 465 470 475 480 Leu Ser Lys Asn Ser Lys Pro Ile His Thr Thr Ile Leu Asn Pro His 485 490 495 Val His Val Ile Gly Glu Asp Ala Ala Cys Ile Ala Tyr Ile Arg Leu 500 505 510 Thr Gln Tyr Ile Asp Gly Gln Gly Arg Pro Arg Thr Ser Gln Ser Glu 515 520 525 Glu Thr Arg Val Trp His Arg Arg Asp Gly Lys Trp Leu Asn Val His 530 535 540 Tyr His Cys Ser Gly Ala Pro Ala Ala Pro Leu Gln 545 550 555 

What which is claimed is:
 1. An isolated nucleic acid molecule encoding a calcium/calmodulin-dependent protein kinase, wherein the nucleic acid molecule consists of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a protein comprising the amino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consisting of SEQ ID NO:1; and (c) a nucleotide sequence consisting of SEQ ID NO:3.
 2. A nucleic acid vector comprising a nucleic acid molecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4. A process for producing a polypeptide comprising SEQ ID NO:2, the process comprising culturing the host cell of claim 3 under conditions sufficient for the production of said polypeptide, and recovering said polypeptide from the host cell culture.
 5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.
 8. A vector according to claims 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the protein of SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence.
 10. An isolated nucleic acid molecule consisting of a nucleotide sequence that is completely complementary to a nucleotide sequence of claim
 1. 